Applied Biosystems 7300 Getting Started Guide

Chapter 3 Performing Reverse Transcription
Guidelines for Preparing RNA
18 Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide
Notes
Primer Extended on mRNA
5′
3′
Reverse
Primer
5′
Synthesis of 1st cDNA strand
cDNA
Oligo d(T) or random hexamer
3′
5′
cDNA
Guidelines for Preparing RNA
Isolating RNA 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.
Quality of RNA The total RNA you use for RQ experiments should:
• Have an A
260/280
greater than 1.9
• 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.
Adjusting the
Starting
Concentration of
Total RNA
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 Part Number
ABI P
RISM
®
6100 Nucleic Acid PrepStation 6100-01
Tem p us
™
Blood RNA Tubes
(For collection, stabilization, and isolation of total RNA in whole blood
for gene expression analysis using the 6100 PrepStation)
4342972
Isolation of Total RNA from Whole Blood and from Cells Isolated from
Whole Blood Protocol
4332809
Tempus
™
Blood RNA Tube and Large Volume Consumables Protocol 4345218
Tissue RNA Isolation: Isolation of Total RNA from Plant and Animal
Tissue Protocol
4330252
Isolation of Total RNA from Cultural Cells: ABI PRISM
™
6700 Automated
Nucleic Acid Workstation or 6100 Nucleic Acid PrepStation Protocol
4330254
NucPrep
®
Chemistry: Isolation of Genomic DNA from Animal and Plant
Tissue Protocol
4333959
ABI P
RISM
®
TransPrep Chemistry: Purification of gDNA from Filtrates
Obtained After the Isolation of RNA from Homogenized Animal or Plant
Tissue Samples Protocol
4326965
BloodPrep
™
Chemistry: DNA Isolation from Fresh and Frozen Blood,
Tissue Culture Cells, and Buccal Swabs on the ABI PRISM
®
6100
Nucleic Acid PrepStation Quick Reference Card
4345297

Applied Biosystems 7300 Specifications

General

Brand	Applied Biosystems
Model	7300
Category	Laboratory Equipment
Language	English