FGRS: protocol

 

RTPCR (Reverse Transcription, PCR)

Last Updated

February 12, 2010 7:04 AM


Authored By: Michael Gonzales


Introduction


Reverse Transcription is the method by which we can obtain cDNA from our mRNA templates.  A cDNA library is the complementary DNA that is reverse transcribed from the expressed mRNA. We build such libraries to identify the gene expression in terms of the DNA. Upon obtaining a cDNA library, the sample can then be amplified via PCR to identify mutations and polymorphisms in the sequences and to measure gene expression on low levels of RNA. The objective of this lab, however, is to transcribe the mRNA into cDNA.


Total RNA


You will need to first prepare Total RNA in order to do RT.  Do this now.


Suspend your final total RNA pellet using nanopure water; Pipette the water up and down several times until you see the entire RNA pellet dissolve.  Obtain the concentration of your RNA by taking a sample on the NanoDrop. You’re going to need this concentration to determine the volume of RNA for your reaction.


Reverse Transcription


Each of these components is found in the Superscript II First Strand Synthesis System for RTPCR box in the -20ºC.


  1. 1.Transfer 1 to 5 µg your prepared total RNA into a sterile tube.

    If you have 5 µg then use 5 µg for this reaction.  Do not use more than 5 µg.

  2. 2.RNA Mixture - Add the following components to your RNA:

    1 µL        oligo dT
    1 µL        10 mM dNTPs

    Add autoclaved or nanopure water to bring the volume up to 10 µL.

  3. 3.Heat the mixture to 65ºC for 5 minutes.

  4. 4.Quickly chill the sample on ice for 2 minutes.

  5. 5.Microcentrifuge briefly to bring the solution to the bottom of the tube.

  6. 6.Leave the RNA Mixture on ice while preparing the Reaction Mixture.

  7. 7.Reaction Mixture - Add the following to the new and separate tube:

    Very Important:
    Use a new tube - do not add these components to your RNA solution.

    2 µL        10X RT buffer
    4 µL        25 mM MgCl2
    2 µL        0.1M DTT
    1 µL        RNaseOUT

  8. 8.Add the 9 µL of the Reaction Mixture to the RNA Mixture.  Mix gently and do a quick spin to collect the mixture in the tube.

  9. 9.Incubate sample at 42ºC for 2 minutes.

    Be sure to add a small amount of water to the heat block to ensure proper heat transfer during the incubation period.

  10. 10.Add the following to the reaction:

    1 µL        SS II Reverse Transcriptase (keep on ice the entire time it is out of the -20ºC)

  11. 11.Incubate sample at 42ºC for 50 minutes.

  12. 12.Heat inactivate the enzyme at 65ºC to 70ºC for 15 minutes.

  13. 13.Place the sample on ice for 5 minutes. Mix gently and do a quick spin to collect the mixture in the tube.

  14. 14.Add the following to the reaction:

    1 µL        RNase H (keep on ice the entire time it is out of the -20ºC)

  15. 15.Incubate sample at 37ºC for 20 minutes.

  16. 16.Microcentrifuge sample briefly to bring solution to bottom of the tube.

  17. 17.Keep the sample on ice any time it is out from now forward.


You can freeze this reaction in the -20ºC once it is done or go on to the PCR Amplification.


PCR Amplification


Now that you have cDNA you can use PCR primers (as you always have) to amplify specific specific regions.  Keep in mind, however, that this amplification is now telling you not about the genomic content of the cells you harvested but rather the transcription level of specific genes.  In this manner, primers for post-RT PCR amplification are usually designed against specific coding (transcribed) regions of the genome.


See the standard PCR protocol for instructions.


You will use approximately 5 µL of your RT reaction as DNA for your PCR reaction.