Applied Biosystems 7300 Getting Started Guide

Chapter 3 Performing Reverse Transcription
Converting Total RNA to cDNA
20 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
Storing cDNA After cDNA conversion, store all cDNA samples at −15 to −25 ° C. To minimize repeated
freeze-thaw cycles of cDNA, store cDNA samples in aliquots.
CHEMICAL HAZARD. 10 × RT Buffer may cause eye, skin, and
respiratory tract irritation. Read the MSDS, and follow the handling instructions. Wear
appropriate protective eyewear, clothing, and gloves.
Sample Experiment - Standard Plate
For the example experiment, RNA is extracted from the liver, bladder, and kidney tissues of an individual. RNA
concentration is determined spectrophotometrically (using A
260
), and the RNA is diluted to a final concentration of 50 ng/µL.
The RT master mix is prepared as follows, using guidelines from the High Capacity cDNA Archive Kit Protocol:
The cDNA archive plate is then prepared by pipetting into each well:
•50 µL of the RT master mix
•30 µL of nuclease-free water
•20 µL of RNA sample (bringing the total starting amount of RNA to 1 µg per 100 µL reaction)
The RNA is then converted to cDNA using the thermal cycling parameters for two-step RT-PCR, as described in “Thermal
Profile Parameters for RT” on page 19.
The cDNA is stored at −20 ° C until use.
Component µL/Reaction µL/21 Reactions
‡
‡ Each RT reaction is 100 µL (see below). If you need 5 µL cDNA for each of 104 PCR reactions per tissue (see “Creating a
Relative Quantification (RQ) Plate Document” on page 25), you need 6 RT reactions per tissue. Extra volume (enough for one
additional RT reaction per tissue) is included to account for pipetting losses, as well as extra cDNA for archiving.
10✕ Reverse Transcription Buffer 10 210
25✕ dNTPs 4 84
10✕ random primers 10 210
MultiScribe
™
Reverse Transcriptase, 50 U/µL 5 105
Nuclease-free water 21 441
Total per reaction 50 1050
Liver Kidney Bladder
GR2322

Applied Biosystems 7300 Specifications

General

Brand	Applied Biosystems
Model	7300
Category	Laboratory Equipment
Language	English