Sean Lauber:Reverse Transcription using Superscript II
Using superscript II (Invitrogen CAT# 18064-022). I think superscript III follows the same protocol.
First you need to determine how much cDNA you want to generate. How much will you need for TaqMan? I generally use 10 ng/Taqman reaction and this is done in triplicate. Also considering the number of probes, you will need:
(10 ng cDNA) x (3 replicates) x (# of probes) = total ng of cDNA required
So if I wanted to look at 6 different probes (including the housekeeping gene for relative quantification, using 18S in the Richards' lab), I would need 180 ng of cDNA so I will need 180 ng of RNA (assuming 1:1 conversion). To control for potential differences in efficiency of cDNA synthesis, you must use the same amount of cDNA for each reverse transcription reaction. So you will need to provide 180 ng of RNA for each reverse transcription reaction to control for this. So if you have 4 samples, you need to figure out what volume you will need to give you 180 ng of RNA. As a control for DNA contamination, you must make a NAC (No Amplification Control)
NAC: No Amplification Control (no reverse transcription - assesses for genomic DNA that can act as template) This involves generating an RT reaction that contains everything from a representative sample from a single experiment (experiments done at different times, in different cells, or if the RNA was isolated at a separate time - all need a single representative NAC). So make a tube WITHOUT RTase, while including template and all other reagents. This verifies for the presence of DNA that may be contributing signal.
1. Put appropriate amount of RNA (eg. 180 ng) into a tube
2. Bring the final volume up to 10 μL. If your solution is too dilute you can use a vacuum centrifuge to evaporate excess volume and then bring it up to 10 μL. It is a good idea to prepare all solutions ahead of time.
10 μL RNA (in this example, enough for 180 ng) 1 μL 100 mM dNTPs 1 μL 0.2 μg/μL 6-mer (this acts as the primer for initiating cDNA synthesis)
3. Heat at 65*C for 5 min (use the thermocycler, program "SS2RT")
4. Quickly chill the samples on ice
5. Add the following while the samples are on ice:
4 μL 5x First-strand buffer 2 μL 0.1 M DTT
6. Incubate at 25*C for 2 min before adding:
1.5 μL DEPC-treated water 0.5 μL SSII (NOT in NAC)
7. Incubate at 25*C for 10 min
8. Incubate at 42*C for 50 min
9. Incubate at 70*C for 15 min to inactivate the enzyme
10. Store at -20*C or at 4*C overnight
11. Dilute the sample to a final concentration of 10 ng/5 μL so you'll be adding 10 ng to each Taqman reaction in a 5 μL volume