Page 1 Agilent 2100 Bioanalyzer 2100 Expert User’s Guide...
Page 2: Contents
Should Agilent and the user and written consent from Agilent Technologies, Inc. as have a separate written agreement with warranty terms governed by United States and international copyright laws.
Page 3: Table Of Contents
How to Use this Manual ......................10 Quick Start ..........................14 Looking at 2100 Expert ......................28 Introduction to the Key Features of the 2100 expert............29 Starting 2100 Expert ........................ 31 2100 Expert Work Area ......................32 Closing 2100 Expert ......................... 43 Running and Evaluating Electrophoretic Assays ..............
Page 4 Working with Chip Data and Assays................258 2100 Expert Data Overview ....................259 Handling Assays........................262 Handling Chip Data ........................ 267 Organizing, Backing up, and Archiving 2100 Expert Data ..........269 Importing Data........................271 Exporting Data ........................277 Printing Reports ........................286 Configuring Tables.........................
Page 5: About This Manual
About this Manual Welcome to the User’s Guide for the Agilent 2100 expert software. This manual provides beginners and advanced users with information needed to successfully run electrophoretic and flow cytometric assays with the bioanalyzer. The 2100 expert software allows the control of the bioanalyzer (including diagnostic...
Page 6: In This Manual
Manual” on page 5 gives an overview of the subjects in this manual, and • lists major innovations and improvements of the 2100 expert software. It also lists supplemental literature and shows you how to make efficient use of this manual.
Page 7 “Performing Verifications” on page 325 describes how you can validate your • bioanalyzer system. “Products, Spare Parts, and Accessories” on page 334 lists all parts and • accessories—including reorder numbers—that are required for electrophoretic and flow cytometric measurements. • “Glossary” on page 338 explains terms in context with flow cytometry, electrophoresis, and terms specific to the bioanalyzer software and hardware.
Page 8: Related Documents
Agilent 2100 Bioanalyzer Maintenance and Troubleshooting Guide CD-ROM Publication Number Title G2946-60002 Agilent 2100 Bioanalyzer – How to Use Multimedia CD-ROM Reagent Kit Guides The Reagent Kit Guides give you information on how to perform specific assays, including sample and chip preparation. Publication Number Title...
Page 9 Reagent Kit Guide Cell Fluorescence Assays G2938-90080 Reagent Kit Guide Cell Fluorescence Checkout Kit Application Notes and Technical Notes Application Notes and Technical Notes are available from the Agilent 2100 Bioanalyzer Help Desk or from the lab-on-a-chip web pages: http://www.agilent.com/chem/labonachip Newly Published Documentation Follow this link to see if there is any new documentation: http://www.chem.agilent.com/scripts/Library.asp...
Page 10: How To Use This Manual
How to Use this Manual This manual uses convenient online navigation features and follows certain typographic conventions. Online Navigation Use the interactive bookmarks in this Use Acrobat Reader’s navigation bar frame to move to your desired topic. to move around within a topic. Click here to go to the table of contents.
Page 11 After you have chosen a topic with the bookmarks, use the buttons in Acrobat Reader’s toolbar to move around within the topic. Displays the next page. Returns to the previous view. Click several times to undo more view changes. Displays the previous page. Displays the first page.
Page 12 Emphasis Example: Right-click the ... Term Example: Dot plots show events as dots. Reference to another document Example: Refer to the Agilent 2100 Bioanalyzer Troubleshooting and Maintenance Guide. Blue Cross-reference or hyperlink Examples: “Introduction to the Key Features of the 2100 expert”...
Page 13 Safety Notices, Notes and Tips Safety notices, notes and tips in this document have the following meaning: WA R N I N G A warning notice denotes a hazard. It calls attention to an operating procedure, practice, or the like that, if not correctly performed or adhered to, could result in personal injury or death.
Page 14: Quick Start
The following step-by-step instructions guide you through a measurement with the Agilent 2100 bioanalyzer. Preparing the Agilent 2100 Bioanalyzer Ensure that the proper cartridge is installed in the bioanalyzer. You can identify the installed cartridge by the number engraved at the front.
Page 15 “Loading the Cell Chip into the Bioanalyzer” on page 189, respectively. Switching on the Agilent 2100 Bioanalyzer Make sure the bioanalyzer is connected to line power and connected to the PC. Turn on the line switch at the rear of the instrument.
Page 16 Instrument is not ready for measurement. Switch the instrument off and on again. If the problem persists, call Agilent service. Running a Measurement To start the 2100 expert software on the connected PC, go to your desktop and double-click the following icon: Contents...
Page 17 After startup of the software, you enter the Instrument context: N O T E If you started 2100 expert for the first time after installation, you first need to activate the different software modules with your license keys. See Figure ,“How to Activate...
Page 18 For details on how to set up the bioanalyzer and connect it to a PC, see Agilent 2100 Bioanalyzer Installation and Safety Guide. Make sure that a bioanalyzer has been detected before continuing.
Page 19 Select an assay for the chip run. On the Instrument tab, click the Assays button. – OR – Click the Assays menu. Both will open a menu, allowing you to select an assay for the measurement. Note that you can also select File > Open File to Run. This opens a dialog box allowing you to load either an assay (.xsy) or a chip data file (.xad).
Page 20 Insert the chip in the Agilent 2100 bioanalyzer: Open the lid. The status of the bioanalyzer is updated on the Instrument tab. Check that the cartridge is inserted properly and the chip selector is in the correct position (“1” for electrophoretic assays, “2” for flow cytometric assays).
Page 21 Place the chip into the receptacle. The figure shows this for an electrophoresis chip. Chip The chip fits only one way. Do not force it into place. C A U T IO N Do not force the lid closed. This may damage the cartridge. Carefully close the lid.
Page 22 When the chip is detected, the image on the Instrument tab changes to a chip. If the chip is not detected, open and close the lid again. N O T E If the AutoRun option is active, the chip run starts automatically once a chip has been inserted and the lid has been closed.
Page 23 The number of the sample that is currently being measured is indicated on the information bar: The status bar at the bottom of the window shows the measurement progress for the chip run and the COM port number used for data acquisition. Contents Index...
Page 24 During the chip run, you can do the following: • View the chip data file in the Data context by clicking on the name of the Data File: • Switch to any other context. For example, you can evaluate any chip data file in the Data context, or compare samples in the Comparison context.
Page 25 Viewing the Measurement Results To view the results, switch to the Data context. The data file that has just been generated by your chip run is displayed. The Chip Summary tab shows information on your chip data file, and lets you enter comments regarding chip, samples, and study. Contents Index...
Page 26 In the tree view panel, click any sample name or the ladder. This selects the Electropherogram tab, which displays a data plot of size/migration time versus fluorescence intensity. Peaks have automatically been detected, and their characteristics such as size, concentration, purity, or molarity have been calculated and are shown in the Peak Table at the bottom of the window.
Page 27 What You Can do When the Measurement is Finished When the measurement is finished, you can: • Document your chip run by entering sample names, chip comments, and study information, for example. Evaluate the measurement results by analyzing gel-like images and electropherograms •...
Page 28: Looking At 2100 Expert
Looking at 2100 Expert Before you start running assays on the Agilent 2100 bioanalyzer, you should familiarize yourself with the 2100 expert software: “Introduction to the Key Features of the 2100 expert” on page 29 • • “Starting 2100 Expert” on page 31 “2100 Expert Work...
Page 29: Introduction To The Key Features Of The 2100 Expert
2100 expert provides detailed installation verification and system verification tests on • both the bioanalyzer hardware and software. 2100 expert allows having multiple chip data and/or assay files open at the same time. • 2100 expert features a new integrated data evaluation tool (Comparison context) •...
Page 30 2100 expert has improved instrument control. Two bioanalyzers can be controlled at • one time. It is possible to run measurements as well as diagnostics tests on two bioanalyzers at the same time. 2100 expert has improved printing and reporting functions.
Page 31: Starting 2100 Expert
Starting 2100 Expert To start 2100 expert: Go to your desktop and double-click the following icon: – OR – From the Windows Start menu, select Programs > Agilent 2100 Bioanalyzer > 2100 expert. Contents Index...
Page 32: Expert Work Area
The 2100 expert software has standard elements such as pull-down menus and toolbars, and the main working area, which contains several tabs, some of which have sub-tabs. The 2100 expert work area has the following regions (demonstrated at the Data context): Title Bar...
Page 33 The 2100 expert software can be operated in six modes, called “contexts”: Instrument Context • • Data Context Verification Context • Comparison Context • Assay Context • System Context • N O T E The contexts work independent from each other regarding their data. This means, for example, that you can review data and run measurements at the same time.
Page 34 Using the Contexts bar, the Context menu, or the selection list in the toolbar, you can switch between the contexts: N O T E Menus, toolbars, the tree view, and the main working area (tabs) significantly change when you switch between the contexts. An introduction to the six contexts is given in the following.
Page 35 Instrument Context On startup, 2100 expert enters the Instrument context, where you can run DNA, RNA, protein or cell assays by selecting an assay file and starting the chip run—provided that the bioanalyzer is properly connected, a chip is inserted, and the bioanalyzer lid is closed.
Page 36 N O T E If two bioanalyzers are connected to your PC, you can run both in parallel. During the chip run(s), you can view the status of the bioanalyzer(s): instrument information and real time acquisition data. In the Instrument context, it is also possible to run hardware diagnostic tests on all connected bioanalyzers.
Page 37 Data Context In the Data context, you can view, analyze, and evaluate the results of your chip runs that are presented as • electropherograms, gel-like images, histograms, dot plots, and result tables. export and print the results of your chip runs. •...
Page 38 Verification Context The Verification context is used to run and document qualification tests. For both the bioanalyzer hardware and software tests can be run for: Installation verification • System verification • Contents Index...
Page 39 Verification results are automatically saved in .xvd files. You can re-open .xvd files to review verification results. For details, refer to “Performing Verifications” on page 325. Contents Index...
Page 40 Comparison Context In the Comparison context, you can open multiple electrophoretic chip data files and compare samples of the same assay class (DNA 1000, for example). It is possible to overlay electropherograms recorded by the bioanalyzer and compare the results. Comparison results can be saved in .xac files.
Page 41 Assay Context In the Assay context, you can create your own assays based on Agilent templates by modifying certain data (for example, data analysis setpoints). Assays are stored as .xsy files. Contents Index...
Page 42 System Context In the System context, you can define System Wide Settings for the 2100 expert software such as settings for default • file names and directories, signal colors, or auto export functions view the contents of the System Log Book •...
Page 43: Closing 2100 Expert
Closing 2100 Expert To close the 2100 expert software: From the File menu, select Exit. If a chip run is in progress, the following message appears: Click OK and wait until the chip run is complete. If there are unsaved files open, the following dialog box appears:...
Page 44 By default, all files with unsaved changes are selected. If you click No, 2100 expert quits without saving any changes. If you do not want to quit 2100 expert at this time, click Cancel to return to your 2100 expert session without saving anything.
Page 45: Running And Evaluating Electrophoretic Assays
Running and Evaluating Electrophoretic Assays For running and evaluating electrophoretic assays you need to know the following: • “Principles of Nucleic Acid and Protein Analysis on a Chip” on page 46 “Preparing and Running an Electrophoretic Assay” on page 50 •...
Page 46: Principles Of Nucleic Acid And Protein Analysis On A Chip
Principles of Nucleic Acid and Protein Analysis on a Chip The electrophoretic assays are based on traditional gel electrophoresis principles that have been transferred to a chip format. The chip format dramatically reduces separation time as well as sample and reagent consumption. The system provides automated sizing and quantitation information in a digital format.
Page 47 RNA, or protein/LDS micells are electrophoretically driven by a voltage gradient—similar to slab gel electrophoresis. Because of a constant mass-to-charge ratio and the presence of a sieving polymer matrix, the molecules are separated by size. Smaller fragments are migrating faster than larger ones. Dye molecules intercalate into DNA or RNA strands or protein/LDS micells.
Page 48 The 2100 expert software plots fluorescence intensity versus size/migration time and produces an electropherogram for each sample: Contents Index...
Page 49 The data can also be displayed as a densitometry plot, creating a gel-like image: Contents Index...
Page 50: Preparing And Running An Electrophoretic Assay
Preparing and Running an Electrophoretic Assay An electrophoretic chip run requires the following steps: Switch on the Agilent 2100 bioanalyzer and start the 2100 expert software. “Starting 2100 Expert” on page 31. Select an electrophoretic assay. “Selecting an Electrophoretic Assay for a Chip Run”...
Page 51 Selecting an Electrophoretic Assay for a Chip Run To select an assay: Switch to the Instrument context. In the Tree View Panel, select the bioanalyzer you want to use. In the upper left of the Instrument tab, an icon shows the status of the bioanalyzer. You should see one of the following icons (lid open/closed), indicating that the bioanalyzer is detected by the system: If you do not see one of these icons, check that the bioanalyzer is switched on and...
Page 52 If you need additional help, please refer to the Agilent 2100 Bioanalyzer Maintenance and Troubleshooting Guide. Select an assay for the chip run. On the Instrument tab, click the Assay button. – OR – Click the Assays menu. Both will open the Assays menu, allowing you to select an assay from the submenus.
Page 53 Select a Destination for the chip data file (.xad) that will be generated as the result of the chip run. You can also specify a custom File Prefix for this file. Under Data Acquisition Parameters, enter the number of samples you want to have measured.
Page 54 Preparing Samples, Reagents, and Chips for Electrophoretic Assays Before you can load a chip, you have to prepare the samples and reagents. To find out how to prepare the samples and reagents, refer to the various Reagent Kit Guides available for each LabChip kit. Please refer to these documents for further information and analytical specifications.
Page 55 Essential Measurement Practices (Electrophoretic Assays) General: WA R N I N G Wear hand and eye protection and follow good laboratory practices when preparing and handling reagents and samples. WA R N I N G No data is available addressing the mutagenicity or toxicity of the dye/DMSO reagent.
Page 56 • Use a new syringe and electrode cleaner with each new LabChip kit. Do not touch the Agilent 2100 bioanalyzer during a chip run and never place it on a • vibrating ground. Keep all reagents and reagent mixes refrigerated at 4 °C when not in use.
Page 57 Protein Assays: • Store Protein sample buffer at -20 °C upon arrival. Keep the vial in use at 4 °C to avoid freeze-thaw cycles. Allow the dye concentrate to equilibrate to room temperature for 20 minutes before • use, to make sure the DMSO is completely thawed. Protect the dye from light during that time.
Page 58 Loading the Electrophoresis Chip into the Bioanalyzer The Agilent 2100 bioanalyzer uses different cartridges for electrophoretic and flow cytometric assays. For electrophoretic measurements, the electrode cartridge is required. The electrode cartridge contains 16 electrodes that fit into the wells of DNA, RNA, and Protein chips.
Page 59 If the bioanalyzer is set up for flow cytometric assays, but you want to run electrophoretic assays, proceed as follows: Open the lid and pull down the metal locking lever into the open position as shown in the figure below. Metal lever in open position...
Page 60 Metal lever C A U T IO N Do not touch the electrodes while the cartridge is in the Agilent 2100 bioanalyzer. The electrodes and the high voltage power supplies can be damaged. Push the metal front of the cartridge to ensure a tight connection.
Page 61 To load the prepared chip into the Agilent 2100 bioanalyzer: Open the lid and remove any chip. Adjust the chip selector to position “1” as shown in the following figure. To avoid using incompatible chips and cartridges, a chip selector is installed in the bioanalyzer.
Page 62 C A U T IO N Do not force the chip selector handle when a chip is inserted in the bioanalyzer. Place the prepared chip into the receptacle. The chip fits only one way. Do not force it into place. Chip Chip selector in position “1”...
Page 63 When the chip is detected, the image on the Instrument tab changes to a chip. If the chip is not detected, open and close the lid again. N O T E The displayed image depends on the assay selcted in the software, not the type of chip inserted.
Page 64 Running an Electrophoretic Assay N O T E You can stop a chip run at any time, for example, if errors occurred or if you are not satisfied with the quality of the measurement results that you can observe during the chip run.
Page 65 The number of the sample that is currently being measured is indicated on the information bar: The status bar at the bottom of the screen shows the measurement progress for the chip run and the COM port number used for data acquisition. Contents Index...
Page 66 During the chip run, you can do the following: • View the chip data file in the Data context by clicking on the name of the Data File: Switch to any other context. For example, you can evaluate any chip data file in the Data •...
Page 67 Stopping a Chip Run You can stop a chip run at any time, for example, • if the quality of the measurement results does not meet your expectations, if, for example, after three samples you already have the information you desired and •...
Page 68 N O T E Data acquisition of the current sample will be aborted. The following message appears: Click Yes to stop the chip run. When the chip run is aborted, you can: • Switch to the Data context, where you can view, analyze, and evaluate the results (if any) of your chip run (see “Displaying the Measurement Results (Electrophoresis)”...
Page 69 Entering Chip, Sample, and Study Information During or after a chip run, you can document the run by entering information on chip, samples, and study. In the Data context, select the Chip Summary tab. On the Sample Information sub-tab, you can enter additional information such as sample names and comments.
Page 70 N O T E You may find some input fields already filled in, because chip, sample, and study information are taken over from the base assay or chip data file. Contents Index...
Page 71 From the File menu, select Save. T I P You can import chip, sample, and study information from .txt or .csv files. This is especially helpful and time-saving, if you already have documented a similar chip run in another chip data file. Refer to “Importing Chip, Sample, and Study Information”...
Page 72 Displaying the Measurement Results (Electrophoresis) You can view the measurement results of an electrophoretic chip run as electropherograms or gel-like images. • You can display the electropherograms either one sample at a time, or all samples at the same time to get an overview of the chip run, for example, to see the progress of a reaction.
Page 73 How to Switch Between Single View and Grid View (Electropherograms) To switch between single view and grid view: From the Electropherogram menu, select View Single Sample or View All Samples. – OR – Click the View Single Sample or View All Samples button on the Electropherogram toolbar.
Page 74 How to Navigate Through the Samples At any time—even during a chip run—you can scroll through all samples—either in electropherogram or gel view. To navigate through samples using the Tree View Panel: If the tree view is not visible, select View > Tree View. The tree view panel appears to the left of the tabs, and shows all chip data and assay files as nodes.
Page 75 To browse through samples: From the Electropherogram or Gel menu, select Next Sample or Previous Sample. – OR – Click the Next Sample or Previous Sample button in the toolbar. To switch between electropherogram and gel view: Click the Electropherogram or Gel tab to display the results of the selected sample as an electropherogram or as a gel-like image.
Page 76 To zoom into an electropherogram: From the Electropherogram menu, select Graph Mode > Zoom (default setting). Position the mouse pointer in the electropherogram. Click and hold down the left mouse button. The mouse pointer changes its shape to a magnifying glass Drag the mouse.
Page 77 To pan and scale an electropherogram: From the Electropherogram menu, select Graph Mode > Pan or Scale. Position the mouse pointer in the electropherogram. Click and hold down the left mouse button. The mouse pointer changes its shape to a double-arrow or to a double crosshair. Drag the mouse.
Page 78 To display data points in an electropherogram: From the Electropherogram menu, select Show Data Points or click the button in the toolbar. Data points used to generate the graph are now shown as bullets. Data points are 0.05 seconds apart. To remove the gray-to-white gradient from the background of an electropherogram: From the Electropherogram menu, select Show Gradient.
Page 79 Then perform the cleaning procedure to ensure that the electrodes are clean (i.e., no residues left from the previous assay). The cleaning procedures are described in detail in the appropriate Reagent Kit Guide and in the Agilent 2100 Bioanalyzer Maintenance and Troubleshooting Guide.
Page 80 Good Practices Empty and refill the electrode cleaner at regular intervals (e.g., every five assays). • • The electrode cleaner can be used for 25 assays. C A U T IO N Never use a cloth to clean the electrodes. Electrostatic discharge could damage the high-voltage power supplies.
Page 81: Analyzing And Evaluating The Results Of An Electrophoretic Assay
Analyzing and Evaluating the Results of an Electrophoretic Assay The purpose of electrophoretic assays is to separate sample components and determine their size, concentration, purity, or molarity. Results for a particular sample are calculated after all data for that sample has been read. The steps in data analysis differ depending on the type of assay in use: “Data Analysis: DNA”...
Page 82 Data Analysis: DNA The data analysis process for DNA assays consists of the following steps: Raw data is read and stored by the system for all of the individual samples. The data is filtered and the resulting electropherograms of all samples are plotted. You can change the settings of the data analysis after the run and reanalyze your data.
Page 83 A sizing ladder (see the following example electropherogram), which is a mixture of DNA fragments of known sizes, is run first from the ladder well. The concentrations and sizes of the individual base pairs are preset in the assay and cannot be changed. Contents Index...
Page 84 A standard curve of migration time versus DNA size is plotted from the DNA sizing ladder by interpolation between the individual DNA fragment size/migration points. The standard curve derived from the data of the ladder well should resemble the one shown below.
Page 85 Two DNA fragments are run with each of the samples, bracketing the DNA sizing range. The “lower marker” and “upper marker” are internal standards used to align the ladder data with data from the sample wells. The figure below shows an example of assigned marker peaks in a sample well.
Page 86 If the check box Rest. Digest on the Chip Summary Tab is enabled, the 2100 expert software flags peaks that may have co-migrated:...
Page 87 Data Analysis: RNA and Cy5-Labeled Nucleic Acids The data analysis process for RNA and the Cy5-labeled nucleic acids assays consists of the following steps: Raw data is read and stored by the system for all of the individual samples. The data is filtered and the resulting electropherograms of all samples are plotted. You can change the settings of the data analysis after the run and reanalyze your data.
Page 88 N O T E Peak ratios for the RNA ladder may vary from one batch of RNA 6000 ladder to the next. Assay performance will not be affected by this variation. For the Eukaryote or Prokaryote Total RNA assay, the RNA fragments (either 18S and 28S for eukaryotic RNA or 16S and 23S for prokaryotic RNA) are detected.
Page 89 Alignment of RNA Samples The marker solution that is part of each RNA LabChip kit, contains a 50 bp DNA fragment. This fragment is used as lower marker to align all samples. By default the RNA alignment and the subtraction of the lower marker are enabled for RNA Nano assays.
Page 90 RNA samples. The RIN extension automatically assigns an integrity number to a eukaryote total RNA sample analyzed on the Agilent 2100 bioanalyzer. Using this tool, sample integrity is no longer determined by the ratio of the ribosomal bands alone, but by the entire electrophoretic trace of the RNA sample, including the presence or absence of degradation products.
Page 91 The computation of the RIN is part of data analysis for total RNA samples. The computed RNA integrity number is shown on the Results sub-tab of the Gel or Electropherogram tab of the Data context. It is also included in XML export files and in printed reports. Contents Index...
Page 92 Until now, the computation of the RIN has only been validated for eukaryote total RNA Nano samples. The 2100 expert software also calculates the RIN for prokaryote total RNA samples and for the RNA 6000 Pico assay. Be aware that for these samples, the RIN has not been validated in extensive downstream experiments.
Page 93 RIN: 1 RIN: 2 RIN: 3 RIN: 4 Contents Index...
Page 94 RIN: 6 RIN: 5 RIN: 7 RIN: 8 Contents Index...
Page 95 RIN: 9 RIN: 10 Contents Index...
Page 96 Computation of the RNA Integrity Number and Signal Anomalies For the computation or the RNA integrity number, the electropherogram is partitioned into regions as shown in the figure below. The lower marker and the 18S and 28S fragments divide the electropherogram into nine regions: 18S-fragment pre-region inter-region...
Page 97 Anomaly Description Critical? Unexpected baseline signal Unexpected signal in pre-region Unexpected signal in 5S-region Unexpected signal in fast-region Unexpected signal in inter-region Unexpected signal in precursor-region Unexpected signal in post-region Unexpected ribosomal ratio Unexpected sample type Unexpected lower marker (compared to previous well) Two categories of anomalies were introduced, critical and non-critical.
Page 98 If a non-critical anomaly is detected, the RIN can still be computed accurately. Therefore non-critical anomalies are not flagged. Non-critical region anomalies are pre-region anomaly, precursor-region anomaly and post-region anomaly. The electropherogram below gives an example for a non-critical anomaly in the post-region. Contents Index...
Page 99 Troubleshooting the RIN To obtain meaninful and reproducible results, the lower marker and ribosomal bands must be identified correctly. Although the ribosomal fragment identification has been improved, in rare cases (i.e. when analyzing degraded RNA samples) the fragment baseline is not properly set. In this case the user should adjust the baseline settings manually.
Page 100 RNA integrity number after the manual adjustment: RIN=5.7 On details on how to adjust the lower marker and ribosomal bands, please refer to “Changing the Data Analysis” on page 116. Contents Index...
Page 101 Determine the threshold value for the RIN that results in meaningful downstream experiments: Cells/Culture Isolation of total RNA RNA QC via Agilent 2100 bioanalyzer Correlate RIN with downstream experiment and determine threshold RIN for meaningful results (iterative process) Contents Index...
Page 102 Run standard experiment and use RIN to determine if sample integrity is sufficient: Cells/Culture Isolation of total RNA RNA-QC via below Agilent 2100 bioanalyzer threshold RIN above threshold Continue with downstream experiment (microarray, real-time PCR, etc.) Contents Index...
Page 103 RNA Integrity Number Setpoints Various setpoints are available to customize the display of the RIN (RNA Integrity Number). With these setpoints, you can modify the predefined thresholds for anomaly detection. You can find them in the advanced user mode of the setpoint explorer. To adjust the setpoints for a single sample, switch to the Local tab of the setpoint explorer and open the RNA Integrity Number group.
Page 104 Data Analysis: Protein The data analysis process for protein assays consists of the following steps: Raw data is read and stored by the system for all of the individual samples. The data is filtered and the resulting electropherograms of all samples are plotted. You can change the settings of the data analysis after the run and reanalyze your data.
Page 105 A sizing ladder (see the example electropherogram below), which is a mixture of proteins of different known sizes, is run first from the ladder well. The sizes of the individual proteins are preset as kDa in the assay and cannot be changed. Please note that the concentrations may vary slightly from ladder lot to ladder lot.
Page 106 A standard curve of migration time versus size is plotted from the sizing ladder by interpolation between the individual protein size/migration points. The standard curve derived from the data of the ladder well should resemble the one shown below. Contents Index...
Page 107 Two proteins are run with each of the samples, bracketing the sizing range. The “lower marker” and “upper marker” proteins are internal standards used to align the ladder data with data from the sample wells. The figure below shows an example of assigned marker peaks in a sample well.
Page 108 The standard curve, in conjunction with the markers, is used to calculate protein sizes for each sample from the migration times measured. To calculate the concentration of the individual proteins in all sample wells, the upper marker is applied to the individual sample peaks in all sample wells. N O T E The software allows you to define upper and lower markers yourself.
Page 109 Protein Absolute Quantitation Absolute quantitation is calculated based on the relative concentration of a sample and the user-defined standard and the known concentration of this user-defined standard. For protein samples you can enable the use of calibration for each sample and enter the concentration of the standard protein.
Page 110 The calibration standard should be run in different concentrations to generate a calibration curve. The software will automatically produce this calibration curve based on these inputs to determine the actual concentration of samples within the same chip. In the peak tables of the samples, a remark is added to the observation column to identify the calibration protein and the calibrated proteins: Contents Index...
Page 111 The calibration curve can be displayed by switching to the Calibration Curve sub-tab on the Chip Summary tab. Contents Index...
Page 112 Smear Analysis The 2100 expert software allows to perform a smear analysis for all electrophoresis assays. When the smear analysis is enabled, the software allows you to define regions of interest. These regions are used to define the area of broad peaks and determine their part of the total area.
Page 113 Under Smear Analysis, select the check box Perform Smear Analysis. The Region Table sub-tab is added to the Electropherogram tab. Contents Index...
Page 114 Performing Smear Analysis After enabling the smear analysis in the setpoint explorer, you are able to insert regions of interest in the electropherogram. To do so: Select the Region Table sub-tab in the Electropherogram tab. Right-click the electropherogram and select Add region. A region will be inserted into the electropherogram.
Page 115 In the smear analysis table, you can edit the Region Start Size and Region End Size, for example: Contents Index...
Page 116 Changing the Data Analysis Different sets of parameters (data analysis setpoints) can be changed in the software in order to modify the data evaluation for sample analysis: Filtering parameters • Peak find parameters for all samples/peak height for individual samples •...
Page 117 About the Setpoint Explorer The tool allowing you to modify the data analysis setpoints is the Setpoint Explorer. The setpoint explorer is accessible from: Assay Properties Tab • Electropherogram Tab (Single/Grid View) • Gel Tab • Contents Index...
Page 118 On the Assay Properties tab, the setpoint explorer is always visible and lets you modify setpoints globally (for all samples): Contents Index...
Page 119 To show the setpoint explorer, on the Electropherogram/Gel tab, click the vertical bar on the right edge of the application window: The setpoint explorer appears. For electrophoretic assays, you can modify the setpoints globally, that is, for all samples (Global tab) •...
Page 120 When you try to change any global setpoints where local settings have been applied, the software prompts you as to whether you want to overwrite the local (custom) settings. If you decide to overwrite the custom sample settings, all local settings you made will be discarded.
Page 121 Filtering Setpoints The first step the software takes in analyzing the raw data is to apply data filtering. The following filtering setpoints can be changed: Filter Width Defines the data window, given in seconds, used for averaging. The broader the filter width, the more raw data points are used for averaging.
Page 122 The four integrator setpoints that can be changed are: Slope Threshold The Slope Threshold setpoint determines the difference in the slope that must occur in order for a peak to begin. The inverse of this value is used to determine the peak end. Area Threshold The Area Threshold determines the minimum amount of peak area that must be detected before a peak is recognized.
Page 123 Manually Moving Fragment Start and End Points (RNA and Cy5-Labeled Nucleic Acids) It is also possible to alter the start and end points manually for individual fragments in an RNA or Cy5-labeled nucleic acids assay. The integration borders of detected RNA-fragments are displayed in the Fragment Table sub-tab.
Page 124 T I P The fragment table can be directly edited in the setpoint explorer: N O T E Changing the start or end points of the fragment will change the calculated rRNA ratio. It might be convenient to pause the automatic analysis (Electropherogram > Pause Automatic Analysis) until all changes are done.
Page 125 to ensure a good result even at very low signal-to-noise ratios. Choose a single sample. Two vertical green long-dashed lines indicating the setpoints for the Start and End Times (with the baseline drawn between them) are displayed in the window. Move the cursor over the long-dashed line on the left (Start Time setting) and drag the line to the desired position.
Page 126 Assigning Upper and Lower Marker Peaks For each DNA or protein sample, the upper and lower marker peaks are assigned first and then the data is aligned so that the sample markers match the ladder markers in time, allowing the size and concentration of the sample peaks to be determined. RNA samples are aligned to a lower marker exclusively.
Page 127 In case the 2100 expert software did not detect the lower marker in RNA samples correctly, you are able to manually assign it in the same way. C A U T IO N Excluding a peak or manually setting a peak to be an upper or lower marker may cause errors during analysis.
Page 128 Aligning or Unaligning the Marker Peaks The upper and lower are then aligned to the ladder markers by resampling the sample data in a linear stretch or compression using a point-to-point fit. Data before alignment: Contents Index...
Page 129 Markers aligned to the ladder: If the sample marker peaks are either more than twice as far apart or less than half as far apart as the ladder markers, they are assumed to be the wrong peaks, and analysis of the sample stops, producing the error “Marker peaks not detected”.
Page 130 If the marker peaks found using this calculated method fail to align with those of a sample, the 2100 expert software will use the minimum peak height threshold setting instead (if this value is lower than the value for the marker peak).
Page 131 Manual Integration For DNA and Protein assays, the 2100 expert software allows to manually integrate peaks. Manual integration allows you to move, add or delete peak baselines. T I P To move a peak baseline, point along the vertical line, press the CTRL key and left mouse button.
Page 132 The baseline points become visible as blue or green dots. Highlighted baseline points are labelled green and can be moved either along the vertical line (press CTRL key and left mouse button) or along the signal trace (left mouse button). The blue baseline points are fixed and cannot be moved.
Page 133 Adjust the baseline points as appropriate. T I P To move a peak baseline point along the vertical line, press the CTRL key and the left mouse button. To move a peak baseline point along the signal, press the left mouse button only.
Page 134 Example: Removing peaks To remove peaks: Highlight the Electropherogram tab in the Data context and zoom into the electropherogram to enlarge the peak of interest. Select Electropherogram > Manual Integration to switch off the automatic integration. As an alternative you might click the Manual Integration button in the toolbar.
Page 135 Right-click a baseline-point and select Remove Peak from the context menu. The two baseline points and the connecting line will disappear and the integration results shown in the result and peak tables will be updated: Contents Index...
Page 136 Example: Inserting peak baselines To insert peaks manually: Highlight the Electropherogram tab in the Data context and zoom into the electropherogram to enlarge the peak of interest. Contents Index...
Page 137 Right-click the electropherogram and select Add Peak from the context menu. Contents Index...
Page 138 Two baseline points and the connecting line will appear and the integration results shown in the result and peak tables will be updated. Contents Index...
Page 139 T I P If you want to change several baseline points, deactivate the automatic analysis by clicking the Pause Analysis button in the toolbar. This way, the software will not recalculate the data analysis with every change. Once you have changed all baseline points, click the Pause Analysis button again to activate automatic analysis.
Page 140 Click the Automatic Analysis button to enable the integration again. The integration results in the result and peak tables will change according to the changes done. Contents Index...
Page 141 Reanalyzing a Chip Data File N O T E Occasionally you may wish to open and view or reanalyze a chip data file that was run and saved previously. The raw data values are saved in the data file, along with the analysis settings that were chosen for the run, so that the data can be reanalyzed with different settings.
Page 142 Reassign lower marker • DNA and Protein Assays Only: Exclude peaks from analysis • • Reassign upper/lower markers Alignment or no alignment with marker peaks • Manual integration • Protein Assays Only: • Absolute quantitation T I P When applying modified data analysis setpoints, the software will (by default) immediately recalculate the raw data, which takes some time.
Page 143 Comparing Samples from Different Electrophoretic Chip Runs The 2100 expert software allows you to compare the measurement results of samples from different electrophoretic chip runs. Samples to be compared must be from chip runs of the same assay type. In the Comparison context, you can create comparison files, include samples from different chip runs, and compare the samples by overlaying electropherograms, for example.
Page 144 Select a .xad file from the Select Data Files list to display a list of its samples. Right-click a sample name and select Add Sample to New Comparison File. Contents Index...
Page 145 A new comparison file appears in the upper part of the tree view containing the sample. The sample is selected and its electropherogram is shown. Note that the Electropherogram Tab (Single/Grid View) has the same functionality as in the Data context. Contents Index...
Page 146 You can now add further samples from any of the open .xad files to the comparison file. T I P Double-clicking a sample name in the lower part of the tree view or dragging a sample name into the tree view adds the sample to the comparison file that is currently selected in the upper part of the tree view.
Page 147 You can also remove samples from a comparison file. Right-click the sample name and select Delete Sample from Comparison File. Contents Index...
Page 148 When you have added all your samples, you can select the Comparison Summary Tab which displays information on the comparison file, and lets you enter a comment regarding the comparison. Contents Index...
Page 149 To compare the electropherograms of samples, go to the Electropherogram tab, click Overlaid Samples in the toolbar, and select the samples to be compared. Contents Index...
Page 150 Select the Gel tab to display a comparison of the gel-like images of the samples. Note that the Gel Tab has the same functionality as in the Data context. Contents Index...
Page 151 10From the File menu, select Save to save the comparison file (.xac) under the default name, or select Save As to save it under a new name. The default name is derived from the assay class: “ComparisonFileX [Assay Class].xac” where “X” is an autoincremented number. Example: “ComparisonFile0 Protein 200.xac” N O T E You can re-open comparison files to review the comparison results, and to add/remove samples.
Page 152: Result Flagging
Result Flagging Result flagging can be used to assign a user-defined color code to a sample. This lets you easily identify samples with certain properties immediately after a chip run. The color assignment is carried out by applying a sequence of rules to the measurement results obtained for the sample.
Page 153 Regardless of how you create the result flagging rules, there are two options available for the order in which the rules are applied: In Normal mode, the rules are applied in the given order, and the first matching rule will •...
Page 154 T I P The examples shown in this chapter are taken from the demo assay “Demo Protein 200 Plus.xsy”, that comes with the 2100 expert software. You can find this demo assay in the “..\assays\demo\electrophoresis” subdirectory of the 2100 expert installation folder.
Page 155 Defining Result Flagging Rules The rules can be defined on the Result Flagging tab. This tab is available in the Data context if an electrophoretic chip data (.xad.) file is selected and in the Assay context if an electrophoretic assay (.xsy) file is selected. Contents Index...
Page 156 A result flagging rule consists of the following: Label Expression • An optional description for the rule used to label samples meeting this rule. • If Expression An expression built from predefined functions, variables, and logical operators. Comment • An optional comment for the rule. Color Expression •...
Page 157 Color Indication The results of the result flagging rules is displayed: On the Chip Summary Tab: • The colors in the Result Flagging column show which sample matches which rule. Contents Index...
Page 158 On the Gel Tab: • The spot on top of the lane is colored if the sample matches a result flagging rule. On the small gel image on the Lower Panel: • Contents Index...
Page 159 On the Results tab: • Result Flagging Color: color of the result flagging rule that the current sample matches. Result Flagging Label: label of the result flagging rule that the current sample matches. How to Use the Form Mode The Form Mode provides some pre-defined rules (forms) that you can use to define the result flagging rules to color-code your samples.
Page 160 The Search Fragment with Purity form is displayed. Fragment/ protein list Purity Tolerance Logic operation Labels and color definitions Define the fragment size(s) to be searched for. Define the required purity for the fragment size(s) and the tolerance. If you defined several fragment sizes and want all of these to be present in the flagged samples, select the option All of them must be present.
Page 161 Select the color with which the samples that meet the criteria should be marked. Optionally select the color with which samples that do not meet the criteria should be marked. Apply this rule to the samples by clicking the Apply Result Flagging icon All samples are re-evaluated according to the result flagging rule and displayed with the respective colors.
Page 162 The result label can be any arbitrary text or be a logic expression. Expressions are built up of functions, variables, operators, and values. You can manually type in the expressions. But you can also double-click the items in the Functions, Variables, and Operators lists, to insert them in the respective fields.
Page 163 For example, the rule condition NumberOfPeaks() > 0 marks all samples with peaks. If you want to indicate the actual number of peaks with the color code, you need to enter NumberOfPeaks() in the Rule Color field. Then you define light green for the Minimum Value 1 and dark green for 10 peaks as the Maximum Value.
Page 164 Example: Result Flagging Sample 1 contains 100 µg/ml proteins. The electropherogram shows 2 peaks for 2 different proteins, which could be separated. One peak can be found at 32 kDa (LDH). Sample 2 contains 60 µg/ml proteins and shows 3 peaks. Sample 3 contains 80 µg/ml proteins and shows 5 peaks.
Page 165 For sample 1, rule 1 matches and defines the color. Rule 2 would also match, but is not checked, because the procedure stops at the first match. For sample 2, none of the rules match, if there is no peak at 30 kDa +/- 7%. Therefore, this sample will get the default color.
Page 166: Running And Evaluating Flow Cytometric Assays
Running and Evaluating Flow Cytometric Assays For running and evaluating flow cytometric assays, you need to know the following: • “Principles of Flow Cytometric Measurements” on page 167 “Overview of Flow Cytometric Assays” on page 176 • • “Preparing and Running a Flow Cytometric Assay”...
Page 167: Principles Of Flow Cytometric Measurements
Principles of Flow Cytometric Measurements Besides electrophoretic assays (DNA, RNA, and proteins), the Agilent 2100 bioanalyzer supports flow cytometric assays: First, cells are stained with two fluorescent dyes that correspond to biologically • relevant parameters, as described in the application notes available for each assay.
Page 168 Staining Cells With the 2100 expert software, you can differentiate several properties of a cell. The characteristics that are examined depend on the dye, which binds specifically to a cellular constituent or is metabolized by the cell to generate a fluorescent product. You usually use two dyes with different colors.
Page 169 The following dyes are recommended for use as the red stain: Dye (red fluorescence) Max. Excitation Max. Emission wavelength wavelength CBNF (Carboxynaphthofluorescein, 595 nm 675 nm living cell stain) APC (Allophycocyanin, intra- and 650 nm 660 nm extra cellular antibody staining) Cy5 (labeled Streptavidin and 647 nm 665 nm...
Page 170 Cell Detection with the Agilent 2100 Bioanalyzer LabChip technology allows cell measurements by integrating cell flow, hydrodynamic focusing, and fluorescence detection into a microfluidic chip. A cell suspension can be confined or “pinched” to a portion of a microfluidic channel, causing cells to line up in single file for individual cell detection.
Page 171 (and therefore a specific cell property) and the physical properties of the stain itself. The Agilent 2100 bioanalyzer lets you determine the number of cells characterized by a specific pattern of fluorescence.
Page 172 Generating Histograms 2100 expert counts the events, sorts them and displays them according to their fluorescence intensity in histograms. For each color measured, a histogram displays the number of events related to the fluorescence intensity. A large number of events with a high fluorescence value means that a large number of cells containing the fluorescence dye were detected.
Page 173 In the histograms, the bar chart is replaced by a point-to-point line as shown in the following image. For detailed information, see “Using Histograms for Evaluation” on page 212. Contents Index...
Page 174 Generating Dot Plots Single events can also be displayed related to both fluorescence values, generating a map of dot plots. In dot plot view, the events (cells with a minimum fluorescence intensity) are displayed in a coordinate system (logarithmic axis scaling). Each axis represents a fluorescence color.
Page 175 In predefined assays, the borders of the rectangular region represent the markers defined in the corresponding blue and red histograms. Gate The lower left region of a dot plot usually shows no events, due to the defined peak detection threshold that the fluorescence values must exceed. For detailed information, see “Using Dot Plots for Evaluation”...
Page 176: Overview Of Flow Cytometric Assays
These so-called “setpoints” are stored in assay files (.xsy) and are read by the 2100 expert software before it starts the measurement. 2100 expert supports the following assays based on flow cytometry:...
Page 177 Transfection Viability Transfection Viability analysis as described in the Application Note: siRNA transfection optimization with the Agilent 2100 bioanalyzer (Agilent publication number: 5988-9782EN). This assay enables the automatic calculation of transfection efficiency (TE) in histogram view and viability in transfected cells (ViT) in dot plot view. Required gating directions and regions are provided as example, but can be adjusted.
Page 178 The regions of the dot plots are related to the markers of the histograms. Thus, the results of the dot plots are identical to the results of the histograms. Generic assay This assay has no specific settings and can be used to define individual assays. You can freely add markers or regions, and define the gating direction.
Page 179: Preparing And Running A Flow Cytometric Assay
Preparing and Running a Flow Cytometric Assay A flow cytometric chip run requires the following steps: Switch on the Agilent 2100 bioanalyzer and start the 2100 expert software. Details are given in “Starting 2100 Expert” on page 31. Select a flow cytometric assay.
Page 180 Analyze and evaluate the results: • – “Using Histograms for Evaluation” on page 212 – “Using Dot Plots for Evaluation” on page 233 – “Evaluating Antibody Staining, Apoptosis, and GFP Assays” on page 242 Contents Index...
Page 181 Selecting a Flow Cytometric Assay for a Chip Run To select an assay: Switch to the Instrument context. In the Tree View Panel, select the bioanalyzer you want to use. In the upper left of the Instrument tab, an icon shows the status of the bioanalyzer. You should see one of the following icons (lid open/closed), indicating that the bioanalyzer is detected by the system: If you do not see one of these icons, check that the bioanalyzer is switched on and...
Page 182 If you need additional help, please refer to the Agilent 2100 Bioanalyzer Maintenance and Troubleshooting Guide. Select an assay for the chip run. On the Instrument tab, click the Assay button. – OR – Click the Assays menu. Both will open the Assays menu, allowing you to select an assay from the submenus.
Page 183 Select a Destination for the chip data file (.xad) that will be generated as the result of the chip run. You can also specify a custom File Prefix for this file. If required, change the Data Acquisition Parameters: Enter the number of samples you want to be measured. When preparing the chip (see “Preparing Samples and Chips for Flow Cytometric Assays”...
Page 184 Select the Data Acquisition Mode. Select Default, if you want the measurement time to be set to the default value (240 s/sample). The maximum time is shown in brackets. – OR – Select Fixed time and enter the time in [s] that the measurement of each sample is to take.
Page 185 Preparing Samples and Chips for Flow Cytometric Assays WA R N I N G Several substances such as dyes can have toxic, carcinogenic, or mutagenic potential. Therefore, carefully follow the safety instructions from the dye safety data sheet and the Reagent Kit Guides. Also read the “Essential Measurement Practices (Flow Cytometric Assays)”...
Page 186 Chip Reagents Several reagents have to be added to the chip to prepare it for measurement. The following image shows which reagents have to be filled in which wells. Priming solution Cell buffer Sample 1 – 6 Cell buffer Focusing dye solution Make sure you follow these directions when preparing the sample: The priming solution has to be added first.
Page 187 Essential Measurement Practices (Flow Cytometric Assays) Handle and store all reagents according to the instructions given in the Reagent Kit • Guides. • Avoid sources of dust or other contaminants. Foreign matter in reagents and samples or in the wells of the chip will interfere with assay results. Store all reagent and reagent mixes in the dark and refrigerated at 4 °C when not in use.
Page 188 Prepared chips must be used within 5 minutes. If a chip is not run within 5 minutes, • beads may settle or reagents may evaporate, leading to poor results. • Never touch the instrument lens. Never touch the Agilent 2100 bioanalyzer during a chip run and never place it on a • vibrating ground. Contents Index...
Page 189 Loading the Cell Chip into the Bioanalyzer The Agilent 2100 bioanalyzer uses different cartridges for electrophoretic and flow cytometric assays. For flow cytometric measurements, the pressure cartridge is required. The pressure cartridge contains a tubing and filter assembly that connect to the vacuum pump.
Page 190 Open the lid and pull down the metal locking lever in the open position as shown in the following figure. C A U T IO N Do not touch the electrodes while the cartridge is in the Agilent 2100 bioanalyzer. The electrodes and the high voltage power supplies can be damaged. Metal lever...
Page 191 Gently pull the cartridge out of the lid. C A U T IO N Improper handling of the electrode cartridge will damage it. Always store the electrode cartridge in the provided box. If the pins of the electrode cartridge are bent or misaligned, poor quality results or pre-terminated chip runs will result.
Page 192 To load the prepared chip into the Agilent 2100 bioanalyzer: Open the lid and remove any chip. Adjust the chip selector to position “2” as shown in the following figure. To avoid using incompatible chips and cartridges, a chip selector is installed in the bioanalyzer.
Page 193 C A U T IO N Do not force the chip selector handle when a chip is inserted in the bioanalyzer. Place the prepared chip into the receptacle. The chip fits only one way. Do not use force. Cell chip Chip selector in position “2”...
Page 194 Carefully close the lid. C A U T IO N Do not force the lid closed. This can damage the pressure cartridge. If the lid does not close without force, check that chip is inserted correctly and that the chip selector is at the correct position for this chip type.
Page 195 N O T E If the AutoRun option is active, the chip run starts automatically once a chip has been inserted and the lid has been closed. Contents Index...
Page 196 Running a Flow Cytometric Assay Running a flow cytometric assay in 2100 expert just means pressing a button. N O T E You can stop a chip run at any time, for example, if errors occurred, or if you are not satisfied with the quality of the measurement results, which you can observe during the chip run.
Page 197 The chip run starts. The Dot Plot sub-tab shows single events (cells) as they are detected, displayed as dots. In the coordinate system, the red and blue fluorescence intensity of each event can be read. The name of the currently measured sample is displayed above the graph.
Page 198 The number of the sample that is currently being measured is indicated on the information bar: The status bar at the bottom of the screen shows the measurement progress for the chip run and the COM port number used for data acquisition. During the chip run, you can do the following: •...
Page 199 After the chip run is completed, you can: • Switch to the Data context, where you can view, analyze, and evaluate the results of your chip run (see “Displaying the Measurement Results (Flow Cytometry)” on page 205 and “Analyzing and Evaluating the Results of a Flow Cytometric Assay”...
Page 200 Stopping a Chip Run You can stop a chip run at any time, for example, • if the quality of the measurement results does not meet your expectations, if, for example, after three samples you already have the information you desired and •...
Page 201 N O T E Data acquisition of the current sample will be aborted. The following message appears: Click Yes to stop the chip run. When the chip run is aborted, you can: Switch to the Data context, where you can view, analyze, and evaluate the results (if •...
Page 202 Entering Chip, Sample, and Study Information During or after a chip run, you can document the run by entering information on chip, samples, and study. In the Data context, select the Chip Summary tab. On the Sample Information sub-tab, you can enter additional information for samples, such as names for blue and red stain.
Page 203 N O T E You may find some input fields already filled in, because chip, sample, and study information are taken over from the base assay or chip data file. Contents Index...
Page 204 From the File menu, select Save. T I P You can import chip, sample, and study information from .txt or .csv files. This is especially helpful and time-saving, if you already have documented a similar chip run in another chip data file. Refer to “Importing Chip, Sample, and Study Information”...
Page 205 Displaying the Measurement Results (Flow Cytometry) You can view the measurement results of a flow cytometric chip run as histograms or dot plots. • You can display the histograms/dot plots either one sample at a time, or all samples at the same time to get an overview of the chip run, for example, to see the progress of a reaction.
Page 206 How to Switch Between Single View and Grid View To switch between single view and grid view: From the Histogram or Dot Plot menu, select Single View or Grid View. – OR – Click the Single View or Grid View button on the histogram/dot plot toolbar.
Page 207 How to Navigate Through the Samples At any time—even during a chip run—you can scroll though all samples—either in histogram or dot plot view. To navigate through samples using the Tree View Panel: If the tree view is not visible, select View > Tree View. The Tree View Panel appears to the left of the tabs, and shows all chip data and assay files as nodes.
Page 208 How to Change the Display of Histograms and Dot Plots In single view, it is possible to change the display of histograms and dot plots. In histograms and dot plots you can: • zoom (enlarge or reduce using the mouse) the graphs to display details, for example. put a color gradient on the background of the graphs.
Page 209 You can perform several zoom steps in a row. When you have zoomed a histogram or dot plot, the Undo Zoom and Undo All buttons are enabled. To undo one zoom step: Click the Undo Zoom button or double-click in the histogram or dot plot. To undo all zoom steps: Click the Undo Zoom All button.
Page 210 To display data points in histograms: From the Histogram menu, select Show Data Points. All events are shown as bullets. To put a color gradient on the background of a histogram or dot plot: From the Histogram or Dot Plot menu, select Gradient. –...
Page 211: Analyzing And Evaluating The Results Of A Flow Cytometric Assay
Analyzing and Evaluating the Results of a Flow Cytometric Assay You can analyze and evaluate result data of flow cytometric assays using either the dot plot or the histogram view. In both views, you can evaluate the detected cells by defining areas of interest.
Page 212 Using Histograms for Evaluation Histograms are graphical representations of the measurement results, where the number of events (cells) is mapped to the Y axis and their fluorescence values to the X axis. The resulting curves show the frequency distribution of the events in relation to their fluorescence intensity values, as shown in the following image.
Page 213 Markers Markers are used to define a range of fluorescence intensity values in a histogram. The upper and lower limits of the range are displayed as vertical lines, as shown in the following image. Lower limit of the marker Upper limit of the marker Events (cells) of interest Low intensity The numerical values for each defined marker are displayed in a separate row in the...
Page 214 Gating Gating is used to restrict the number of events that are evaluated by gating out (filtering) events that do not have the fluorescence values set by a marker. For example, by gating on a blue marker, you can exclude all events with low blue fluorescence (allowing you, for example, to gate out dead cells, unbound dye and debris).
Page 215 Gating from red to blue uses the red histogram to define the subset by a marker • (GFP assay). N O T E Predefined assays have a fixed gating direction, while assays of type Generic have a variable gating direction. Contents Index...
Page 216 The following figures illustrate gating from blue to red. The two histograms display all measured events in the blue histogram and in the red histogram without gating. In this case, you cannot see which cells fluoresce only in the blue and which fluoresce only in the red. All events that show red fluorescence All events that show...
Page 217 By setting a marker on the blue histogram, you can define the blue fluorescence range that must be met for a cell to be considered for the red histogram. You use the gating on the blue histogram to define a subset for the red histogram. Subset of the events, defined by the marker Events of the subset that...
Page 218 The red histogram displays now only cells with blue and red fluorescence within the marker. To evaluate this subset, you can set a marker in the red histogram. This second marker filters out all cells that do not have fluorescence in this range. Events with high blue fluorescence Events that show both high...
Page 219 The following image shows two histograms with a gating direction from blue to red (left to right) of an apoptosis assay. The blue histogram shows calcein fluorescence, which indicates living or dead cells (high fluorescence value means living cells). The red histogram shows the subpopulation of living cells with annexin V fluorescence indicating apoptosis (high fluorescence value means the cell is apoptotic).
Page 220 How to Insert a Marker in a Histogram A marker is shown as two vertical lines that define a region of fluorescence values. It is used to select a subset of events according to this fluorescence region. N O T E You can insert markers only in generic assays.
Page 221 You can remove markers that you do not need any more: Click one of the vertical lines in the histogram to select the marker. The lines of the selected marker are displayed bold. T I P You can also click the corresponding row in the result table to select the marker. Click the Delete Marker button to remove the marker.
Page 222 How to Configure Markers You can change the color, name, and the upper and lower limits of the marker: Double-click the desired marker. – OR – Right-click the corresponding row in the result table and select Configure Marker from the context menu. –...
Page 223 Enter an Upper Value (right vertical line). N O T E The lower and upper values must be within the range of 0.01 – 10000 relative fluorescence units. Click the Color button to open the Color dialog box and select a color. Click OK.
Page 224 How to Move the Upper and Lower Limits of Markers You can change the position of both marker lines by dragging them with the mouse: Position the mouse pointer on a marker line. The mouse pointer changes its shape to a hand. Drag the line to the desired position.
Page 225 How to Copy Markers to All Histograms Once a marker is defined, you can copy it in the histograms of all samples (generic assays only): Select the marker in the histogram or in the result table. The Insert the selected marker into all histograms button is now enabled.
Page 226 To set the gating direction: Select the marker in the red or blue histogram you want to use as a gate for the other histogram. The corresponding gating button in the toolbar is now enabled. Click to set the gating direction. –...
Page 227 How to Overlay Histograms You can compare samples by overlaying their gated histograms. This is useful, for example, if you want to see the progress of a reaction or if one sample is used as reference. Overlaying histograms might also be helpful for adjusting the marker position. You can overlay all measured samples.
Page 228 To overlay all samples: Click the Overlaid Samples button to open a drop-down list. Select All Samples to overlay the histogram curves of all samples. To remove histograms from the overlay: Select the sample that contains the overlaid histograms. Click the Overlaid Sample button to open the drop-down list.
Page 229 How to Set Signal Colors for Overlaid Histograms You can use the Graph Settings tab in the Options dialog box to configure the signal colors (colors of curves in histograms): Select Tools > Options. Click the Graph Settings tab to bring it to the front. To configure the signal color: Click the colored square corresponding to the signal.
Page 230 Displaying the Results of Histogram Evaluations The calculated results are displayed in result tables, one table below each histogram. Markers, gates, several statistical values, and the %-values of events are shown in the result tables. Each marker you insert in the histogram gets its own row. Note that you can only use one marker for gating.
Page 231 The content of the result tables depends on the gating direction. The histogram that is used for gating can display the following results: Marker All events – this row shows the data for all measured events, for example, for all living and dead cells. The following rows show the data for the subset of cells defined by the inserted marker.
Page 232 The histogram that displays the gated data can show the following data: Marker All events – this row shows the data for all events that pass the gate. The following rows show the data for all events covered by the inserted marker.
Page 233 Using Dot Plots for Evaluation On the Dot Plot tab, cells are displayed as dots, where their red fluorescence intensity is mapped on the Y axis and their blue fluorescence intensity is mapped on the X axis. N O T E The lower left region of the dot plot area may show no events, because of the threshold for event detection.
Page 234 How to Add Regions to Dot Plots (Generic Assay only) You can draw regions in dot plots of generic assays. If there are regions already defined in other samples, you can copy these regions in the dot plot of the current sample. To draw a new region: Click the Insert Region button in the toolbar.
Page 235 To insert an existing region: Select the sample where you want to insert an existing region from another sample and click Insert existing region The Insert Region dialog box appears. Select the region that you want to insert and click Insert Region. The region is inserted at its predefined position.
Page 236 How to Configure Regions You can change the color of the region border, edit the region’s name, and define the position and size of the region. To configure a region: Double-click the border of the region that you want to configure. –...
Page 237 Enter fluorescence values for the left, right, bottom, and top side of the rectangle to define position and size of the region. These values correspond to the upper and lower marker limits of the blue and red histograms. Click the ... button next to the color square to open the Color dialog box, and select a color for the region border.
Page 238 How to Change Position and Size of a Region You can change the size and position of regions to restrict the number of included events. You can work graphically with the mouse or enter the values in the Configure Region dialog box.
Page 239 To change size and position numerically: Double-click the region to open the Configure Region dialog box. Enter fluorescence values for the left, right, bottom, and top side of the rectangle to define position and size of the region. These values correspond to the upper and lower marker limits of the blue and red histograms.
Page 240 Click No to create new regions that are not “connected”. The region will be inserted in the dot plots of all other samples. When the properties of the region are changed, the changes affect only the selected sample. The region is copied to all samples of the assay. How to Work with Gates in Dot Plots You can insert gates only in generic assays.
Page 241 Displaying the Results of Regions The measurement results and calculations for regions are displayed in the result table below the dot plot. In predefined assays, only one region is available, while for generic assays, dot plots can have as many regions as you like. The following values are displayed: Region The default region All Events is always displayed in the first row and...
Page 242 Evaluating Antibody Staining, Apoptosis, and GFP Assays With the 2100 expert software, several predefined assays are supplied. You should only use each assay for the specific experiment for which it was developed. For example, you have to use the read dye for detection of apoptosis (calcein and Cy5, for example): “Evaluating Antibody Staining...
Page 243 You can use a blue dye like calcein to detect whether or not the cells are living, or like SYTO 16 to stain the nucleic acids of all cells. For detailed information, refer to the application note Detecting Cell Surface and Intracellular Proteins with the Agilent 2100 Bioanalyzer by Antibody Staining.
Page 244 Histogram Evaluation The blue histogram is used for gating. High fluorescence in the blue histogram means that the cells are living (if a life-indicating dye is used). Low fluorescence means the cells are dead. If you use a nucleic acid dye, you cannot distinguish between living and dead cells, you can only count all measured cells.
Page 245 When using the calcein marker in the blue histogram for gating, only living cells are considered for building the histogram of the red dye. High red fluorescence values indicate living cells with bound antibodies, low red fluorescence values living cells without bound antibodies.
Page 246 Dot plot evaluation If you switch to the Dot Plot tab, one region is displayed in the dot plot. The red fluorescence values of the region are related to the marker in the red histogram, the blue fluorescence values to the marker in the blue histogram. As in the histogram evaluation, high blue fluorescence and high red fluorescence mean living cells with bound antibodies.
Page 247 The results of the dot plot evaluation are numerically displayed in the result table: Events covered by the region All measured events Amount of living cells in relation to all measured cells Amount of living cells with high antibody binding in relation to all living cells Evaluating Apoptosis Assays The apoptosis assay can be used to examine how many apoptotic cells are within a living cell population.
Page 248 For detailed information, refer to the application note Apoptosis Detection by Annexin V and Active Caspase 3 with the Agilent 2100 Bioanalyzer. Apoptotic cells In apoptotic cells, phosphatidylserine is no longer confined to the inner leaflet of the plasma membrane bilayer.
Page 249 High fluorescence value indicates living cells Low fluorescence value indicates dead cells The values are displayed in the result table, each histogram has its own table: All measured events All events in relation to the blue marker (here calcein) Living cells in relation to all measured cells (high calcein fluorescence) When using the calcein marker in the blue histogram for gating, only the living cells are considered for building the red histogram.
Page 250 High fluorescence value indicates living apoptotic cells Low fluorescence value indicates living non-apoptotic cells Amount of the living cells in relation to all measured events Percentage of all cells with high red fluorescence selected by the red marker Amount of living cells with high red fluorescence in relation to the amount of living cells Contents Index...
Page 251 Dot plot evaluation If you switch to the Dot Plot tab, one region is displayed in the dot plot. The red fluorescence values of the region are related to the marker in the red histogram, the blue fluorescence values to the marker in the blue histogram. As in the histogram evaluation, high blue fluorescence and high red fluorescence represent living cells with annexin V binding.
Page 252 The results of the dot plot evaluation are displayed in the result table. All measured events Events covered by the region Amount of living cells with high red fluorescence in relation to the amount of all cells Amount of living cells with high red fluorescence in relation to the amount of living cells Contents Index...
Page 253 For detailed information on GFP assays, refer to the application note Monitoring transfection efficiency by green fluorescent protein (GFP) detection with the Agilent 2100 Bioanalyzer. For a detailed description on how to evaluate the results using histograms and regions, refer to “Using Histograms for...
Page 254 High fluorescence value is associated with living cells Low fluorescence value indicates dead cells The values are displayed in the result table, each histogram has its own table. All events related to the red marker (here CBNF) All measured events After gating by using the red histogram, in the blue histogram only CBNF-stained cells are displayed.
Page 255 High fluorescence value indicates GFP-producing cells Amount of the CBNF containing cells in relation to all measured cells Percentage of GFP containing cells in relation to all measured cells Percentage of GFP containg cells in relation to the cells gated by the CBNF marker Contents Index...
Page 256 Dot plot evaluation If you switch to the Dot Plot tab, one region is displayed in the dot plot. The red fluorescence values of the region are related to the marker in the red histogram, the blue fluorescence values to the marker in the blue histogram. Corresponding to the histogram evaluation, high blue fluorescence and high red fluorescence indicate living GFP-producing cells.
Page 257 The results of the dot plot evaluation are displayed in the result table. Events covered by the region All measured events Amount of cells with high CBNF fluorescence and high GFP fluorescence in relation to all measured events Amount of cells with high GFP fluorescence in relation to the amount of CBNF-stained cells.
Page 258: Working With Chip Data And Assays
Working with Chip Data and Assays You can make efficient use of the chip and assay data generated by the 2100 expert software, if you know the following fundamentals and operating techniques: • “2100 Expert Data Overview” on page 259 “Handling...
Page 259: Expert Data Overview
2100 Expert Data Overview The 2100 expert software manages data in the following different formats: • Assay files (.xsy) Chip data files (.xad) • • Comparison files (.xac) Verification result files (.xvd) • Diagnostics result files (. xdy) • •...
Page 260 Ladder table and peak table (electrophoretic assays only) • Result flagging rules (electrophoretic assays only) • Chip data files Chip data files (.xad) contain the following information: Measurement results • After each chip run, the measurement results—also called “raw data”—are automatically saved in a new chip data file.
Page 261 The files are stored in the “..\validation” subfolder of the 2100 expert installation directory. For each verification run, an .xvd file is generated. Date and time of the verification run are included in the file name. Example: “Verification_23-05-2005_10-28-40.xvd”.
Page 262: Handling Assays
Handling Assays Predefined Assays Predefined assays are provided with 2100 expert. They are meant and prepared for measurements using the available LabChip kits. Predefined assays such as Apoptosis or DNA 1000 are write-protected. Although you can open predefined .xsy files and edit some of their properties, you cannot save any changes under the original file name.
Page 263 The Assays menu is dynamically built from the structure and contents of the “..\assays” subdirectory of the 2100 expert installation folder. T I P You can add items to the Assays menu by placing assay (.xsy) files—your own assays, for example—in subdirectories of the “..\assays”...
Page 264 How to Create a Custom Assay To create a custom assay: Switch to the Assay context. From the Assays menu, select an assay. – OR – Select File > Open and open an assay (.xsy) file. The file appears in the Tree View Panel. N O T E If you want to create a new flow cytometric assay with free gating direction or with more than one marker or region, open and modify the assay “Generic.xsy”.
Page 265 Tab. N O T E The study description is stored in the 2100 expert system file. Altering the study description of an assay will not affect the entries in the data files that were previously generated from this assay. To update this information in the data files, too, they must be opened, and the study must be assigned again.
Page 266 – For electrophoretic assays, define or modify flagging rules on the Result Flagging Tab. From the File menu select Save to save the method with the current name or Save as to save it with a new name. Contents Index...
Page 267: Handling Chip Data
Directories” on page 300). Modifying and saving chip data files 2100 expert allows to re-open chip data files, reanalyze them using different evaluation parameters and store the new results. You can save modifications either to the original file (File > Save) or under a new file (File > Save As).
Page 268 The benefit of opening chip data files as read-only is to prohibit you or other users from making changes that would alter the file in any way. Because the 2100 expert software allows you to open chip data files, modify data, and save them, you may prefer to ensure that the original parameters that were used to create the file are not altered.
Page 269: Organizing, Backing Up, And Archiving 2100 Expert Data
Organizing, Backing up, and Archiving 2100 Expert Data As you begin to work with the 2100 expert software, it is good practice to organize your files. If you are not the only user of the bioanalyzer, creating a directory within which to save your files is recommended;...
Page 270 It is a good idea to periodically archive your files to a CD/DVD to remove them from your hard disk. Depending on the amount of hard disk space available to the 2100 expert software, you may need to clear space on your hard drive to ensure that you will have enough room to save upcoming chip run data.
Page 271: Importing Data
Importing Data When working with assay (.xsy) or chip data (.xad) files, you enter specific information that you may want to reuse. To support the reuse of data, 2100 expert has the following import capabilities: “Importing Bioanalyzer Files” on page 272 •...
Page 272 Importing Bioanalyzer Files You can import data, assay and method files that were generated with other Agilent 2100 bioanalyzer systems. You can even import data and assay files from the older Bio Sizing and Cell Fluorescence software applications. To import assay files: Switch to the Assay context.
Page 273 The imported file appears in the Tree View Panel, and the electropherogram grid view shows an overview of all samples. Upon importing, the file gets converted to a new 2100 expert chip data file (.xad). Importing Data Analysis Setpoints You can import data analysis setpoints from other assay (.xsy) or chip data (.xad) files of the same type.
Page 274 N O T E For flow cytometry files, the import will delete all existing markers and regions in the current file, and change the current assay to a Generic assay. A message box appears that prompts you to confirm this change. Click Yes.
Page 275 Importing Chip, Sample, and Study Information On the Sample Information and Study Information sub-tabs of the Chip Summary tab, you can enter names and comments regarding chip, samples, and study. The information you enter here may be very similar for further chip runs or other assays. Once you have entered the information, you can export it into a separate file (see “Exporting Chip Run Data”...
Page 276 Importing Result Flagging Rules You can import result flagging rules into electrophoretic assay (.xsy) or chip data (.xad) files. Result flagging rules can be stored in .xml files (see “Exporting Result Flagging Rules” on page 285). To import result flagging rules: Open an electrophoretic assay or chip data file in the respective context.
Page 277: Exporting Data
Exporting Data 2100 expert allows you to export the results of your chip runs in a variety of formats. The exported data can be used for further evaluation with other applications, such as text ® processors, graphic tools, MS Excel , or flow cytometry applications.
Page 278 Exporting Chip Run Data To export chip run data: Switch to the Data context. In the Tree View Panel, select a chip data (.xad) .file or load a file. From the File menu, select Export. If you selected an electrophoretic chip data file, the Electrophoresis Export Options dialog box appears.
Page 279 N O T E Keep in mind that exporting a chip data file can require up to 20 MB of disk space. In particular, exporting electropherograms and gel-like images as .tif or .bmp files may take up a lot of disk space. Click Export.
Page 280 Exporting Chip Run Data Automatically N O T E Keep in mind that exporting a chip data file can require up to 20 MB of disk space. In particular, exporting electropherograms and gel-like images as .tif or .bmp files may take up a lot of disk space.
Page 281 Exporting Tables You can export: Result tables, peak tables, fragment tables, and ladder tables as .csv files or .xls files. • • Log book tables as .html or .txt files. To export a result table, peak table, fragment table, or ladder table: On the Assay Properties, Electropherogram, Gel, Histogram, or Dot Plot tab, right-click the heading row of a table.
Page 282 Exporting Graphs You can export graphs as individual graphic files. This applies to all graphs that can be displayed in 2100 expert such as electropherograms or dot plots. To export a graph: Right-click the graph, and select the appropriate entry (e.g. Save Gel or Save Electropherogram) from the context menu.
Page 283 You can also copy tables (or parts of tables) into the clipboard. This applies to most of the tables that can be displayed in 2100 expert, such as result tables or log book tables. To copy a graph or table into the clipboard: Right-click the graph or table (region).
Page 284 You can now switch to a word processing, spreadsheet, graphics, or other application, and paste the graph or table there. Exporting Chip, Sample, and Study Information On the Sample Information and Study Information sub-tabs of the Chip Summary tab, you can enter names and comments regarding chip, samples, and study.
Page 285 Exporting Result Flagging Rules You can export result flagging rules for reuse in other electrophoretic assay (.xsy) or chip data (.xad) files (see “Importing Result Flagging Rules” on page 276). Result flagging rules are stored in .xml files. To export result flagging rules: Open the electrophoretic assay or chip data file with the desired result flagging rules in the respective context.
Page 286: Printing Reports
When printing manually, a preview function allows you to view the printout before starting the print job. The 2100 expert program can also be set to print customized chip run reports automatically at the end of the run. These reports can be set up to contain different information (settings for the manual and automatic print functions are maintained separately).
Page 287 How to Print a Chip Run Report The following information can be included in a chip run report: You can always include: • – Run summary—general data about the assay, and sample information. – Assay details—complete list of data analysis setpoints. –...
Page 288 To print a report: Switch to the Data context. In the Tree View Panel select the chip data (.xad) file you want to generate a report of. From the File menu select Print. Depending on the file type different dialog boxes appear. You generally have the following possibilities: –...
Page 289 N O T E Your selections here are separate from the Auto Print selections (they do not affect each other). Both are used by default the next time you print (even after restarting the program). Use the Page Setup and Printer buttons to access system dialog boxes, allowing you to select a printer, and specify the print medium and page layout.
Page 290 The following example shows the “Run Summary” part of an RNA chip run report. Contents Index...
Page 291 How to Turn on and Configure Automatic Printing of Chip Run Reports A report can be automatically printed on a printer or generated as a file at the end of each chip run. Saving reports as files can be helpful for documentation purposes. To enable and configure automatic printing: Switch to the System context.
Page 292 – In the Print Item section, select the options that are to be included in the report. – In the Save To File section, you can redirect the automatic printouts to .pdf and .html files. Note that no print output is generated if you select the PDF and/or HTML option. –...
Page 293: Configuring Tables
Configuring Tables 2100 expert uses various tables to present data: Result tables • • Peak tables Fragment tables • • Log book tables In some cases, you might want to reorganize the way the data is presented. To do so, you can hide or show columns, change the column sequence, and adapt the table height.
Page 294 Showing and Hiding Columns To add the Aligned Migration Time column to the table: Right-click the heading row of a table and select Configure Columns from the context menu. The Configure Columns dialog box opens. Contents Index...
Page 295 Move any desired column headers from the Available list to the Displayed list. Configure the order of the column headers in the Displayed list by using the Up and Down buttons. Click OK. A new column Aligned Migration Time is inserted in the table: Contents Index...
Page 296 Changing the Column Sequence T I P You can set the column sequence also using the Up and Down buttons in the Configure Columns dialog box. To change the column sequence of a table: Position the mouse pointer on a column header. Click and hold the left mouse button, and drag the header cell to the desired position.
Page 297 Changing the Table Height You can customize the view by changing the height of the table. To increase or reduce the table height: Position the mouse pointer above the heading row of the table and move it upwards until the cursor’s shape changes to a double arrow. Click and hold the left mouse button and drag up or down.
Page 298 In this example, the Peak Table freed screen space for the gel-like image above the table: Contents Index...
Page 299: Administering System Functions
Administering System Functions The 2100 expert software provides the following configuration options and system functions: Default data file names and directories can be specified. Also, settings such as for • automatic printing or automatic data export can be set up. See “Configuring...
Page 300: Configuring 2100 Expert
Configuring 2100 expert The available options for configuring the 2100 expert software can be found in the System context on the System Wide Settings tab. How to Specify Data File Names and Directories The measurement results are stored automatically when the chip run is complete. To make it easier for you to identify the chip data files, you can configure an automatic naming scheme for the files.
Page 301 Option Meaning Prefix Inserts an arbitrary string to identify the data file. This string can be modified. The default file prefix is “2100 expert”. Assay Class Inserts the assay class in the file name. Examples: “DNA1000”, “GFP”, “Apoptosis”. Contents...
Page 302 Use the Browse button to select a directory or click Reset if you want to use the “..\Data” directory under the 2100 expert installation directory. Optionally, you can select the check box Create Daily Subdirectories if you want daily subdirectories to be created.
Page 303 How to Set Run and Result Options You can select several options such as to pause the analysis on setpoint changes, the maximum log file size, or the graph colors. To set the Run and Result options: Switch to the System context and select the System Wide Settings tab. Select Run and Result in the tree navigation.
Page 304 – select Auto Run to activate the automatic start of a chip run once the lid of the Agilent 2100 bioanalyzer is closed and a chip suiting the selected assay is detected. – select Auto Print to enable the automatic report printing function.
Page 305 How to Set Auto Export Options To define auto export options: Switch to the System context and select the System Wide Settings tab. Select Auto Export in the tree navigation. The Auto Export screen becomes visible: Activate the Auto Export check box, if you want a data file to be exported automatically after every chip run.
Page 306 How to Activate Software Licenses By installing the 2100 expert software you have also installed a license administration tool. This tool is used to activate the different software modules. The following licenses can be ordered separately: 2100 electrophoresis license •...
Page 307 To activate an additional software license: Select Registration from the Help menu to open the License Administration Tool window. Switch to the Add License tab. In the Select Product field, the Agilent 2100 Bioanalyzer must be selected. Contents Index...
Page 308 The licensed software modules are now activated and can be used. N O T E If you added the license key to activate the security pack, the 2100 expert software closes and the secured file area will be set up. Follow the instructions displayed in the different pages of the setup wizard.
Page 309 N O T E Store your license keys in a secure place and make sure you do not lose them. Contents Index...
Page 310: Using Log Books
Using Log Books 2100 expert provides several log books to document all relevant actions and changes. Due to requirements of data integrity and data security, none of the log books can be cleared. Run Logs The run log books can be found in the following contexts as sub-tabs of the Log Book tab: •...
Page 311 The system log book can be found in the System context as a sub-tab of the Log Book tab. It includes start-up and shut-down events of the 2100 expert software, and, for example, errors or problems with the connected bioanalyzers.
Page 312 The system log book is saved in config/SystemFile.xml. The log book entries can be exported from this file. How to Change the Display of the Log Books To sort a log book table: Click the column header you want to sort the table by. The log book table is sorted by the entries in the selected column in ascending order.
Page 313 To remove the filter from a log book table: In the Log Book toolbar, click Reset T I P You can hide/show any of the log table columns, and re-sort the columns by right-clicking the table and selecting Columns from the context menu. Contents Index...
Page 314 How to Search the Log Book You can search the various log books for any string. To search the Log Book: In the Log Book toolbar, click Find The Find dialog box appears. Enter a search string in the Find What field. Use the Column selection list to specify whether you want to search all columns or a particular column only.
Page 315 To continue the search, click Find Next. Contents Index...
Page 316: Running Instrument Diagnostics
Running Instrument Diagnostics 2100 expert provides several tests to check proper functioning of the bioanalyzer hardware. You should perform the tests on a regular basis, or if incorrect measurements occur. You can test the following: Generic bioanalyzer tests, which can be run with both types of cartridges (electrode or •...
Page 317 Generic Bioanalyzer Tests Diagnostics Test Purpose Electronics Test Verifies proper functioning of all electronic boards in the bioanalyzer. Fan Test Checks if the fan is running at the appropriate speed. Lid Sensor Test Verifies proper operation of the lid sensor, ensuring that the laser and LED are off when the lid is open.
Page 318 Electrode Cartridge Tests Diagnostics Test Purpose HV Stability and Tests high voltage accuracy and stability of all 16 high voltage Accuracy Test power supplies and the high voltage controller. Unused chip (DNA, RNA, or protein) required. HV Accuracy Test Check of channel-reference diode in transmission direction. (On-Load) Short Circuit Test Checks for instrument leak currents using an empty chip.
Page 319 Pressure Cartridge Tests Diagnostics Test Purpose Pressure Offset Test The vacuum system of the pressure cartridge consists of a pump and the corresponding tubes. This test calibrates the pressure sensors to zero. Pressure Control Test Checks if the bioanalyzer is able to hold the working pressure of -140 mbar.
Page 320 Test Chips Depending on your bioanalyzer setup (electrophoresis or flow cytometry), different test chips are required to run some of the diagnostics tests. Test chip kits are part of the bioanalyzer electrophoresis set (G2947CA) and flow cytometry set (G2948CA): Test Chip Kit for Electrophoresis Assays (reorder no. G2938-68100) Test Chip Comment Quantity...
Page 321: How To Run Instrument Diagnostics Tests
The 2100 expert software will generate an error message if a wrong cartridge type is detected for the selected assay. To run the selected test please insert the requested cartridge type (see “Loading the Electrophoresis Chip into the...
Page 322 Select the tests you want to run: – Select the Apply check boxes to select single tests. – Click Select All to select all available tests. – Click Unselect All to deselect all tests. Click Start. Contents Index...
Page 323 Follow the instructions given by the 2100 expert software. For example, exchange the cartridge, or put a test chip in the receptacle of the bioanalyzer when requested by the software. All selected tests are performed. Contents Index...
Page 324 If any test failed, redo the test. If failures still persist, contact Agilent service. The results of diagnostics tests are stored in .xdy files in the 2100 expert installation folder under “..\diagnosis”. If tests fail, send the .xdy files to the Agilent service.
Page 325: Performing Verifications
Performing Verifications To ensure a validated Agilent 2100 bioanalyzer system, verification steps have to be performed at installation and operation level. 2100 expert allows for detailed installation verification and system verification on both the bioanalyzer hardware and software. Each verification comprises a series of tests and measurements that you can run and document in the Verification context of the 2100 expert software.
Page 326 System Verification System verification proves that the bioanalyzer system is suitable for its intended use, that is, that it will function according to its operational specifications in the selected environment. System verification should be performed: • at first use of the instrument, after relocating the instrument, •...
Page 327 Under Configure 2100 Bioanalyzer HW Test Chips, enter the test chips you will use for this verification: Contents Index...
Page 328 In the Tree View Panel, navigate to the test category you want to execute. Select the category via Installation/System Verification – Software/Hardware – PC name/Bioanalyzer name – Test Category. N O T E To execute hardware tests (HW branch) the bioanalyzer must be properly connected and switched on.
Page 329 To start the selected tests, click Start button in the toolbar. The Save As dialog box appears. Specify a name and location for the verification results file (.xvd) and click Save. The selected tests are executed. Contents Index...
Page 330 If a test fails, you can Repeat test execution, Abort the verification run, or skip the current test and Continue with the next test: 10After all tests have been executed the following message appears: 11Click OK. Contents Index...
Page 331 12The Status column shows which of the tests have been run successfully, which have failed, and which have mixed results with multiple executions. Contents Index...
Page 332 13To view details on test execution, select the Results tab. 14You can now navigate to other test categories and execute additional verification tests. Contents Index...
Page 333 15When you close the verification result file (File > Close), try to switch to another context, or exit 2100 expert, the following message appears: If you select No, you return to the Verification context and can run further verification tests.
Page 334: Products, Spare Parts, And Accessories
Products, Spare Parts, and Accessories To buy the following products, spare parts and accessories for the Agilent 2100 bioanalyzer, please refer to the Agilent Online Store: http://www.agilent.com/home/buyonline.html Bundles • G2940CA – Agilent 2100 bioanalyzer desktop system Includes Agilent 2100 bioanalyzer, HP Compaq desktop PC, color printer, system software, vortexer, and accessories.
Page 335 Software and Services G2946CA – Agilent 2100 expert software upgrade • Software package for upgrade to the latest 2100 bioanalyzer system software revision. G2949CA – Agilent 2100 expert security pack • Additional services for Installation Qualification (IQ) and Operation Qualification/Performance Verification (OQ/PV) as well as assay consulting are available and can be ordered separately.
Page 336 G2938-68200 – Test Chip Kit for Flow Cytometric Assays Comprises 1 Cell Autofocus Chip G2938-81605 – RS 232 cable • Communication cable PC – Agilent 2100 Bioanalyzer 2110-0007 – Fuse • • 5042-1398 – Adjustable Clip for use as spare part for the chip priming station 5065-4401 –...
Page 337 5065-9966 – Vortex Mixer Adapter for IKA vortexer • 5065-9951 – Electrode Cleaner Box • Contains 7 electrode cleaners • G2946-60002 – Agilent 2100 bioanalyzer How to Use CD-ROM Contains videos showing the chip preparation for all assays and the hardware maintenance Contents Index...
Page 338: Glossary
An assay is a solution with defined chip, chemicals, instrument methods, data analysis, data output settings and data display settings. ASY file In Bio Sizing electrophoretic assays were stored as .asy files. 2100 expert can import .asy files. See also XSY file. Contents...
Page 339 Audit Trail Audit trails are available in the 2100 expert software only with the security pack installed. They are used to record the activities of the logged-in users and cannot be modified. The audit trails as well as log books are subject to data protection. Only authorized users are allowed to inspect them.
Page 340 The figure below shows baselines established for DNA assay peaks. Peaks for DNA and protein assays are determined on a peak-by-peak basis (the overall baseline is shown). Contents Index...
Page 341 The figure below shows baselines established for Total RNA assay fragments. Total RNA fragments are determined on a peak-by-peak basis and an overall baseline is shown from the start to end time. Contents Index...
Page 342 The figure below shows baselines established for an mRNA assay. mRNA fragments are determined on a peak-by-peak basis and an overall baseline is shown from the start to end time. Contents Index...
Page 343 N O T E With RNA assays, you can move the lines marking the start and end points for data analysis (shown by the long-dashed vertical green lines) which will adjust the entire baseline for calculation of the area of the total sample. Baseline Plateau This setpoint (found in the setpoint explorer) rejects brief, low slope areas such as at peaks and between non-baseline-resolved peaks.
Page 344 CAD file In Cell Fluorescence flow cytometric chip runs were stored as .cad files. 2100 expert can import .cad files. See also XAD file. Center Point After locating a start point, the peak find algorithm looks for the first negative slope value and saves the previous point as the center.
Page 345 Data Filtering The first step 2100 expert takes in analyzing raw data is to apply data filtering. Data filtering is done by means of a polynomial “filter” that is applied to the raw data. The setting for the Polynomial Order in the setpoint explorer determines the amount of data to be applied: the smaller the number, the more data that is applied and the more filtering that takes place.
Page 346 Electrokinetic forces Electrokinetic forces are used to move, switch and separate the samples. Active control over voltage gradients directs the movement of materials using the phenomenon of electrophoretic flow. Electroosmotic Flow A phenomenon that results from an electrical double layer formed by ions in the fluid and surface electrical charges immobilized on the capillary walls.
Page 347 End Point The peak find algorithm looks for a leveling off when the value of the slope is less than the value set for the slope threshold. This is considered to be the end point of the peak. With RNA assays, individual peak end times can be moved manually by dragging the diamond-shaped end points shown in the single-well display.
Page 348 Firmware The firmware is a program to control the hardware of the Agilent 2100 bioanalyzer. It is downloaded from your computer to the Agilent 2100 bioanalyzer and controls, among others, data transfer or the measurement procedures. Flow Cytometry A method to detect cells with certain properties. In a continuous stream, stained cells pass through a light beam.
Page 349 center point value (local baseline) must be greater than the Height Threshold value. This setting is chosen in the setpoint explorer. Histogram Histograms are bar charts to display, for example, a frequency distribution. HTML file HTML (Hyper Text Markup Language) is the authoring language used to create documents on the World Wide Web.
Page 350 Lab-on-a-chip The generic term for a microfluidic product, signifying a chemical process or material movement taking place on a microchip. In contrast to analysis in a standard laboratory that relies on human intervention at several stages to manipulate or observe samples and record results, the self-contained lab-on-a-chip represents an almost hands-free technology.
Page 351 The lower marker is the same as the first peak found in the DNA ladder. Method Methods are available in the 2100 expert software only with the security pack installed. A method is referred to as an electrophoretic or flow cytometric assay with additional information stored to it.
Page 352 capability of liquid transfer, separation, dilution, reactions and more • Molarity where: Molarity is measured in nanomoles per liter (nmol/l) Concentration is measured in nanograms per microliter (ng/µL) Size is measured in base pairs (bp) is the molecular weight of a single base pair Miniaturized laboratories on a microchip Expression used to describe lab-on-a-chip technology.
Page 353 PDF file PDF (Portable Document Format) is a file format created by Adobe Systems Incorporated that preserves all of the fonts, formatting, colors, and graphics of any source document, regardless of the software and computer platform used to create it. Peak Baseline A local peak baseline is calculated for each peak.
Page 354 Peak Filter Width The Peak Filter Width setpoint determines the minimum amount of time that must elapse before a peak is recognized. Peak Height The value at the center point of the peak minus the local baseline start value. Point-to-Point Fit This curve fit is composed of line segments between each pair of data points that are used to interpolate data between those points.
Page 355 Polynomial Filter The first step 2100 expert takes in analyzing the raw data is to apply data filtering. Data filtering is done by means of a polynomial “filter” that is applied to the raw data. Priming Station Consists of a chip holder that has a syringe mounted on the lid that seals over the chip.
Page 356 Serial port The serial ports (COM ports) are used to connect your computer with the Agilent 2100 bioanalyzer. The number of available ports depends on the computer you use. Signature Signatures are available in the 2100 expert software only with the security pack installed.
Page 357 Start Time This setting determines the time after which the first peak or fragment will be located (any peaks appearing before this time are ignored). In RNA and Protein assays, the start time is shown on the single view display as a long-dashed vertical green line (note that this is true for protein assays when analysis is on;...
Page 358 This includes steps such as the execution of methods, result reviews, and the final approval. The workflow definition is part of the methods and is available in the 2100 expert software only with the security pack installed.
Page 359 XAD file 2100 expert chip data file. The files contain raw data, assay information, data analysis setpoints, information on chip, samples and study, and the run log information. XAC file 2100 expert comparison file. XLS file Microsoft Excel spreadsheet file.
Page 360 2100 expert verification result file. The files contain results of verification tests regarding the bioanalyzer hardware and software. xvd. files are stored in the “..\validation” subfolder of the 2100 expert installation directory. For each verification run, an .xvd file is generated.
Page 361 Zero Baseline All electropherograms produced with the bioanalyzer show some amount of background fluorescence. By default, the 2100 expert software enables the zero baseline function. Enabling this setting offsets the graphs shown for the individual wells but does not affect analysis.
Page 362 To remove the zeroing, disable the Zero Baseline box in the setpoint explorer (baseline calculation under Global and Advanced setting). None-Zero Baseline Contents Index...
Page 363 Index Accessories, 334 Calcein, 168 Adding regions, 234 Capillary electrophoresis, 352 Agilent Online Store, 334 Cartridges, 58, 189 Alignment, 128 CBNF, 169 Antibody staining, 243 Cell detection, 170 APC, 169 Chip reagents, 186 Apoptosis assays, 247 Color Assay setpoints, 262 overlaid histograms, 229 Assays Comparing samples, 143...
Page 364 Data analysis setpoints, 262 Histogram Data files, 260 generating, 172 Data points, 76, 208 overlaying, 227 Documentation, related, 8 Dot Plot generating, 174 Dyes, 168 Inserting marker, 220 Inserting peaks, 136 Installation verification, 325 Inverse pipetting, 187 Electrode cartridge, 58 Electrodes, 60, 190 Electrokinetic forces, 346 Electroosmotic Flow, 346...
Page 365 Molecular weight, 352 importing rules, 276 Multi channel mode, 73, 206 Result table histogram values, 230 regions, 241 RIN, 90 Navigation, 74, 207 RNA integrity number, 90 Run log, 310 Opening assays, 265 Overlay histograms, 227 Scale color and scale, 229 overlaid histograms, 229 Setpoint explorer, 117 Setpoints, 262...
Page 366 Zoom Dot Plot, 76, 208 Histogram, 76, 208 Undo, 77, 209 Contents Index...

Agilent Technologies 2100 User Manual

About this Manual

Quick Start

Looking at 2100 Expert

Running and Evaluating Electrophoretic Assays

Running and Evaluating Flow Cytometric Assays

Working with Chip Data and Assays

Administering System Functions

Running Instrument Diagnostics

Performing Verifications

Products, Spare Parts, and Accessories

Glossary

Quick Links

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Summary of Contents for Agilent Technologies 2100

Table of Contents