Guidelines For Preparing Rna; Chapter 3 Performing Reverse Transcription - Applied Biosystems 7500 Getting Started Manual

Chapter 3 Performing Reverse Transcription

Primer Extended on mRNA
5' 3'
5' cDNA
Reverse

Guidelines for Preparing RNA

Primer
Oligo d(T) or random hexamer
Synthesis of 1st cDNA strand
5' cDNA
3'
Guidelines for Preparing RNA
Isolating RNA
Quality of RNA
Adjusting the
Starting
Concentration of
Total RNA
Notes
18
Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide
Applied Biosystems supplies several instrument systems and chemistries for RNA
isolation from a variety of starting materials, such as blood, tissue, cell cultures, and
plant material.
®
ABI P 6100 Nucleic Acid PrepStation
RISM
™
Tempus Blood RNA Tubes
(For collection, stabilization, and isolation of total RNA in whole blood
for gene expression analysis using the 6100 PrepStation)
Isolation of Total RNA from Whole Blood and from Cells Isolated from
Whole Blood Protocol
™
Tempus Blood RNA Tube and Large Volume Consumables Protocol
Tissue RNA Isolation: Isolation of Total RNA from Plant and Animal
Tissue Protocol
Isolation of Total RNA from Cultural Cells: ABI P
Nucleic Acid Workstation or 6100 Nucleic Acid PrepStation Protocol
®
NucPrep Chemistry: Isolation of Genomic DNA from Animal and Plant
Tissue Protocol
®
ABI P TransPrep Chemistry: Purification of gDNA from Filtrates
RISM
Obtained After the Isolation of RNA from Homogenized Animal or Plant
Tissue Samples Protocol
™
BloodPrep Chemistry: DNA Isolation from Fresh and Frozen Blood,
Tissue Culture Cells, and Buccal Swabs on the ABI P
Nucleic Acid PrepStation Quick Reference Card
The total RNA you use for RQ experiments should:
• Have an A greater than 1.9
260/280
• Be intact when visualized by gel electrophoresis
• Not contain RT or PCR inhibitors
The High Capacity cDNA Archive Kit Protocol (PN 4312169) contains additional
guidelines for preparing the RNA template.
The High Capacity cDNA Archive Kit is optimized to convert 0.1 to 10 µg of total RNA
to cDNA. Convert enough total RNA so that the final concentration of total RNA
converted to cDNA is 10 to 100 ng in 5 µL for each 50-µL PCR reaction.
Products
™
6700 Automated
RISM
®
RISM
Part Number
6100-01
4342972
4332809
4345218
4330252
4330254
4333959
4326965
4345297
6100