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Qiagen RNeasy Fibrous Tissue Handbook

Fibrous tissue mini kit
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January 2018
RNeasy
®
Fibrous Tissue
Mini Handbook
For purification of total RNA
from heart, skeletal muscle,
aorta and other fiber-rich
tissues
Sample to
Insight__

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Summary of Contents for Qiagen RNeasy Fibrous Tissue

  • Page 1 January 2018 RNeasy ® Fibrous Tissue Mini Handbook For purification of total RNA from heart, skeletal muscle, aorta and other fiber-rich tissues Sample to Insight__...

  • Page 2: Table Of Contents

    Handling and storing starting material ............. 12 Disrupting and homogenizing starting material ..........13 Protocol: Purification of Total RNA Using the RNeasy Fibrous Tissue Mini Kit ....15 Troubleshooting Guide ..................... 22 Appendix A: General Remarks on Handling RNA ............25 Appendix B: Storage, Quantification, and Determination of Quality of RNA ....

  • Page 3: Handbook Revision History

    Removal of RNeasy Fibrous Midi Kit (cat. no. 75742), including January 2018 associated protocols, general kit information (Table 1 and Table 2) and ordering information. Update into Sample-to-Insight branding and edit into revised style. RNeasy Fibrous Tissue Mini Handbook 01/2018...

  • Page 4: Kit Contents

    * Contains a guanidine salt. Not compatible with disinfectants containing bleach. See page 6 for safety information. † Before using for the first time, add 4 volumes of ethanol (96–100%) as indicated on the bottle to obtain a working solution. RNeasy Fibrous Tissue Mini Handbook 01/2018...

  • Page 5: Storage

    Storage The RNeasy Fibrous Tissue Mini Kit is shipped at ambient temperature and should be stored dry at room temperature (15–25°C). The RNase-Free DNase Set should be stored immediately upon receipt at 2–8°C. All components are stable for at least 9 months under these conditions.

  • Page 6: Safety Information

    For more information, please consult the appropriate safety data sheets (SDSs). These are available online in convenient and compact PDF format at www.qiagen.com/safety where you can find, view and print the SDS for each QIAGEN kit and kit component.

  • Page 7: Introduction

    Introduction The RNeasy Fibrous Tissue Mini Kit is designed for optimal lysis of fiber-rich tissues and purification of high-quality total RNA >200 nt. Principle and procedure Total RNA purification from fibrous tissues, such as skeletal muscle, heart and aorta tissue can be difficult due to the abundance of contractile proteins, connective tissue and collagen.
  • Page 8 Figure 1. RNeasy Fibrous Tissue Mini workflow. RNeasy Fibrous Tissue Mini Handbook 01/2018...

  • Page 9: Equipment And Reagents To Be Supplied By User

    Tissue Reagent (cat. no. 76405) or liquid nitrogen and dry ice 1.5 ml or 2 ml microcentrifuge tubes  Microcentrifuge (with rotor for 2 ml tubes)  * Do not use denatured alcohol, which contains other substances such as methanol or methylethylketone. RNeasy Fibrous Tissue Mini Handbook 01/2018...

  • Page 10: Important Notes

    RNA to the RNeasy Mini spin column membrane, resulting in lower RNA yield and purity. More information on using the correct amount of starting material is given in the protocols. Table 2 (next page) shows expected RNA yields from various sources. RNeasy Fibrous Tissue Mini Handbook 01/2018...
  • Page 11 10–15 * Amounts can vary due to factors such as species and developmental stage. Since the RNeasy Fibrous Tissue Mini Kit enriches for mRNA and other RNA species >200 nucleotides, the total RNA yield does not include 5S rRNA, tRNA and other low-molecular-weight RNAs, which make up 15–20% of total cellular RNA.

  • Page 12: Handling And Storing Starting Material

    After disruption and homogenization in Buffer RLT (lysis buffer), samples can be stored at –90 to –65°C for several months. Storage times differ for different tissue types and have not been established for all possible tissues. RNeasy Fibrous Tissue Mini Handbook 01/2018...

  • Page 13: Disrupting And Homogenizing Starting Material

    For guidelines on using the TissueRuptor II, refer to the TissueRuptor II Handbook. For other rotor–stator homogenizers, refer to suppliers’ guidelines. RNeasy Fibrous Tissue Mini Handbook 01/2018...
  • Page 14 5 mm mean diameter. In this case, we recommend using the RNeasy 96 Universal Tissue Kit (cat. no. 74881), which provides high- throughput RNA purification from all types of tissue – including fiber-rich tissues – in 96-well format. RNeasy Fibrous Tissue Mini Handbook 01/2018...

  • Page 15: Protocol: Purification Of Total Rna Using The Rneasy Fibrous Tissue Mini Kit

    3 mm cube (27 mm ) of most animal tissues weighs 25–35 mg. Important points before starting If using the RNeasy Fibrous Tissue Mini Kit for the first time, read “Important Notes”  (page 10). If working with RNA for the first time, read “Appendix A: General Remarks on Handling ...
  • Page 16 DTT can be stored at room temperature for up to 1 month. Buffer RPE is supplied as a concentrate. Before using for the first time, add 4 volumes of  ethanol (96–100%) as indicated on the bottle to obtain a working solution. RNeasy Fibrous Tissue Mini Handbook 01/2018...
  • Page 17 Note: RNA in harvested tissues is not protected until the tissues are treated with RNAlater RNA Stabilization Reagent, flash-frozen or disrupted and homogenized in step 4. Frozen tissues should not be allowed to thaw during handling. The relevant procedures should be carried out as quickly as possible. RNeasy Fibrous Tissue Mini Handbook 01/2018...
  • Page 18 9. Place the tubes in the TissueLyser Adapter Set 2 x 24. 10. Operate the TissueLyser II for 2 min at 20 Hz. Note: The time depends on the tissue being processed and can be extended until the tissue is completely homogenized. RNeasy Fibrous Tissue Mini Handbook 01/2018...
  • Page 19 17. Add 0.5 volumes (usually 450 µl) of ethanol (96–100%) to the cleared lysate. Mix well by pipetting. Do not centrifuge. Precipitates may be visible after addition of ethanol. This does not affect the procedure. RNeasy Fibrous Tissue Mini Handbook 01/2018...
  • Page 20 15 s at ≥8000 x g (≥10,000 rpm) at 15–25°C. Discard the flow-through.* Reuse the collection tube in step 24. * Flow-through contains Buffer RLT or Buffer RW1 and is therefore not compatible with bleach. See page 6 for safety information. RNeasy Fibrous Tissue Mini Handbook 01/2018...
  • Page 21 RNA yield is >30 µg, there is no need to repeat step 28. If using the eluate from step 27, the RNA yield will be 15–30% less than that obtained using a second volume of RNase-free water, but the final RNA concentration will be higher. RNeasy Fibrous Tissue Mini Handbook 01/2018...

  • Page 22: Troubleshooting Guide

    This troubleshooting guide may be helpful in solving any problems that may arise. For more information, see also the Frequently Asked Questions page at our Technical Support Center: www.qiagen.com/FAQ/FAQList.aspx. The scientists in QIAGEN Technical Services are always happy to answer any questions you may have about either the information and/or protocols in this handbook or sample and assay technologies (for contact information, visit www.qiagen.com).
  • Page 23 RNase-Free DNase Set”, page 33. For real-time, two-step RT-PCR experiments, carry out the RT step using the QuantiTect ® Reverse Transcription Kit (cat. no. 205311), which provides cDNA synthesis with integrated removal of genomic DNA contamination. RNeasy Fibrous Tissue Mini Handbook 01/2018...
  • Page 24 To eliminate any chance of possible ethanol carryover, place the RNeasy Mini spin column in a new 2 ml collection tube and perform the optional 1-minute centrifugation step as described in step 26 of the protocol. RNeasy Fibrous Tissue Mini Handbook 01/2018...

  • Page 25: Appendix A: General Remarks On Handling Rna

    Keep purified RNA on ice when aliquots are pipetted for downstream applications. Disposable plasticware The use of sterile, disposable polypropylene tubes is recommended throughout the procedure. These tubes are generally RNase-free and do not require pretreatment to inactivate RNases. RNeasy Fibrous Tissue Mini Handbook 01/2018...
  • Page 26 (SDSs), available from the product supplier. † Plastics used for some electrophoresis tanks are not resistant to ethanol. Take proper care and check the supplier’s instructions. RNeasy Fibrous Tissue Mini Handbook 01/2018...
  • Page 27 DEPC contamination. * When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more information, consult the appropriate safety data sheets (SDSs), available from the product supplier. RNeasy Fibrous Tissue Mini Handbook 01/2018...

  • Page 28: Appendix B: Storage, Quantification, And Determination Of Quality Of Rna

    30), the ratio between the absorbance values at 260 and 280 nm gives an estimate of RNA purity. * When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more information, consult the appropriate safety data sheets (SDSs), available from the product supplier. RNeasy Fibrous Tissue Mini Handbook 01/2018...
  • Page 29 = 44 µg of RNA * When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more information, consult the appropriate safety data sheets (SDSs), available from the product supplier. RNeasy Fibrous Tissue Mini Handbook 01/2018...
  • Page 30 * Wilfinger, W.W., Mackey, M., and Chomczynski, P. (1997) Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity. BioTechniques 22, 474. † Values up to 2.3 are routinely obtained for pure RNA (in 10 mM Tris·Cl, pH 7.5) with some spectrophotometers. RNeasy Fibrous Tissue Mini Handbook 01/2018...
  • Page 31 (see www.qiagen.com/GeneGlobe). For real-time RT-PCR assays where amplification of genomic DNA cannot be avoided, we recommend using the QuantiNova Reverse Transcription Kit (cat. no. 205411) for reverse transcription. The kit integrates fast cDNA synthesis with rapid removal of genomic DNA contamination.
  • Page 32 RNA Integrity Number (RIN) as a measure of RNA integrity. Ideally, the RIN should be close to 10, but in many cases (particularly with tissue samples), how well the original sample is preserved greatly influences RNA quality. RNeasy Fibrous Tissue Mini Handbook 01/2018...

  • Page 33: Appendix C: Dnase Digestion Of Rna Eluates Using The Rnase-Free Dnase Set

    Procedure 1. Purify RNA according to the standard protocol (page 15), but without performing the on-column DNase digestion described in steps 20–23 (instead, perform the optional wash with Buffer RW1 described in step 20). RNeasy Fibrous Tissue Mini Handbook 01/2018...
  • Page 34 The reaction volumes can be doubled if necessary (to 200 µl final volume). 4. Incubate on the benchtop (15–25°C) for 10 min. 5. Clean up the RNA using the RNeasy Mini Kit (cat. no. 74104). RNeasy Fibrous Tissue Mini Handbook 01/2018...

  • Page 35: Ordering Information

    1.5 ml RNAlater RNA Stabilization Reagent each RNAlater TissueProtect Tubes For stabilization of RNA in 20 x 500 mg 76163 (20 x 5 ml) tissue samples: 20 screw-top tubes containing 5 ml RNAlater RNA Stabilization Reagent each RNeasy Fibrous Tissue Mini Handbook 01/2018...
  • Page 36 235 V, 50/60 Hz (for Australia). For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at www.qiagen.com or can be requested from QIAGEN Technical Services or your local distributor.
  • Page 37 QIAGEN specifically disclaims any other licenses, expressed or implied other than those expressly stated. The purchaser and user of the kit agree not to take or permit anyone else to take any steps that could lead to or facilitate any acts prohibited above. QIAGEN may enforce the prohibitions of this Limited License Agreement in any Court, and shall recover all its investigative and Court costs, including attorney fees, in any action to enforce this Limited License Agreement or any of its intellectual property rights relating to the kit and/or its components.
  • Page 38 Ordering www.qiagen.com/shop | Technical Support support.qiagen.com | Website www.qiagen.com RNeasy Fibrous Tissue Mini Handbook 01/2018 1112114...

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