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Reverse Transcription

Reagents

• SuperScript III from Invitrogen #18080-044.
• Oligo dT from Invitrogen #18418-012.
• RNase OUT from Invotrogen #10777-019.
• Rnase H from USB #P/N 70054Z.
• dNTP from Invotrogen #10297-018 (250 ul each, 100 mM). Order the separate nucleotides and mix then dilute using RNase-free H2O (Supplied in the RNeasy kit) to 10 mM each. Store aliquots into -20C.

1. Measure concentration of total RNA.

2. Calculate the volume for 1 ug RNA, calculate the volume of RNase-free H2O to 11 ul.

3. Prepare two sets of RNase-free PCR tubes (8 strips), one for reverse transcription with transcriptase and the other for control without transcriptase.

4. Add H2O to the tube and 1 ug RNA, the total volume is 11 ul.

5. Add 1 ul Oligo dT and 1 ul dNTP (10 mM each nucleotide). The total volume is 13 ul.

6. Use any PCR machine and set up a program for 65C of 5 min. Run the program with the samples.

7. In the meantime, prepare master mix for transcriptase. For each sample, 4 ul 5X buffer, 1 ul RNase OUT, 1 ul DTT solution, 1 ul SuperScriptIII. For the control, replace SuperscriptIII with 1 ul H2O.

8. Quickly chill the PCR tubes with RNA samples on ice for 1 min.

9. Carefully remove caps and add 7 ul master mix into each samples. For the corresponding control tube, add 7 ul master mix with H2O instead of SuperScriptIII.

10. Using PCR machine and set up a program for 50C of 60 min, then 70C of 15 min and finally keep in 4C. Run the program with the samples.

11. (optional) Add 1 ul RNase H into each sample including the controls and incubate at 37C for 20 min. You can transfer the samples to 1.5 ml eppendorf tubes.

12. The samples are now ready for regular PCR or real-time PCR.

13. Always run the control to see whether there is contamination from DNA.

14. For real-time PCR, I usually dilute the sample by 10 using H2O since there are a lots of proteins (RNase OUT, reverese transcriptase, etc) which may affect PCR reactions.