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.

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

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