Molecular Techniques and Methods

Reverse Transcription-Polymerase Chain Reaction
(RT-PCR)

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

RT-PCR offers a rapid, versatile and extremely sensitive way of analyzing whether a target gene is being expressed and can provide some semi-quantitative information about expression levels.
RT-PCR is an extremely valuable tool when limited material, such as specific differentiated cells, is available.
RT-PCR can be used either to detect specific transcripts by using sequence-specific primers, or can be used to create cDNA libraries by using generic primers such as oligo-dT (dT18) and either random oligonucleotides (pdN6) or 5'-cap specific primers.
RT-PCR is based on the ability of the enzyme reverse transcriptase (an RNA-dependent DNA polymerase) to generate a complementary strand of DNA (first-strand cDNA) using the total RNA or mRNA as a template.




MATERIALS AND SOLUTIONS

Random Hexamer (pdN6) (0.5 ug/ul)

Gene-Specific Primer

Oligo-dT18-22 Primer (0.5 ug/ul)
500 ug Oligo-dT18 in 1 ml of DEPC-H2O




PROCEDURES

1. Optional: RNA in DEPC-H2O may be heated at 70oC for 5 min to eliminate RNA secondary structures and in turn to increase the priming efficiency.
  • Do not heat RNA over 80oC. RNA strand nicks at high temperature (>80oC).
  • Treatment at 70oC is only necessary to synthesize the full length cDNA.


    2. Prepare the Master mix as follow in ice:

    Working solution
    Final Concentration
    10x RTase Buffer
    1x
    10 mM dNTP mix
    1 mM
    5-10 nmole Primer
    (Random Hexamer, Gene-Specific Primer, or Oligo dT18-22)
    50-100 pmole
    RNase Inhibitor
    1-5 U
    Reverse transcriptase
    50-200 U
    DEPC-treated H2O
    -


    3. Add Master mix and RNA to each tubes.

    4. For oligo-dT18 priming: Incubate reactions at 37oC for 30 min, and then 42oC for 30 min.
  • For random primer (pdN6) priming: Incubate reactions at 25oC for 5 min, 37oC for 30 min, and then 42oC for 30 min.

    5. After RT reaction, continue to PCR reaction.




    NOTES

    RNA Preparation:
    The reverse transcriptase reaction can be performed on either total cytoplasmic RNA or on purified mRNA. It is important that no genomic DNA is present, as this will also provide a template for the PCR amplification step. An appropriate control for any contaminating DNA is a control reaction in which the reverse transcriptase step is omitted.
  • An RNase-free DNase I digestion step can be included in the RNA extraction protocol.
  • The primers can be designed to amplify across an intron, thus allowing simple identification of contaminating DNA that will contain the intron while the transcript will not. This means DNA will give rise to a longer product than the RNA transcript. The method is called intron-differential RT-PCR.

    The use of purified mRNA is recommended since this generally gives rise to a higher yield of first-strand cDNA. When analyzing low-abundance transcripts, the use of purified mRNA is important for success since the relative concentration of the target mRNA will be much higher than when using total cytoplasmic RNA.


    First Strand cDNA Synthesis:
    The next step is to copy the mRNA to first-strand cDNA. This is often done using an oligo-dT primer that can anneal to the 3'-poly A tail of eukaryotic mRNAs and allows reverse transcriptase to synthesize cDNA from each mRNA molecule present in the reaction. This can be carried out either using purified mRNA.
    There are two common types of reverse transcriptase: Avian Myeloblastoma Virus (AMV) and Moloney Murine Leukemia Virus (M-MuLV).
  • AMV Reverse Transcriptase - has both 5' to 3' primer-dependent polymerase activity with either RNA or DNA as template and a 3' to 5'-RNase H activity that degrades the RNA portion of the RNA-DNA heteroduplex product of cDNA synthesis.
  • M-MuLV-Reverse Transcriptase - is essentially identical to the AMV RTase but it can only use RNA as a template.


    The PCR Reaction:
    Appropriate upstream and downstream primers are used and can either be specific to the target gene or for cDNA library construction. Due to the single-stranded nature of the first-strand cDNA, the early cycles of the PCR involve linear amplification as the first strand can only act as template for one of the primers. Exponential amplification from both primers occurs once sufficient copies of the second strand have been generated.




    KIT INFORMATION




    REFERENCES

  • Franz, O, Brudhhaus, I, Roeder, T (1999) Verification of differential gene transcription using virtual northern blotting. Nucleic Acids Res. 27: 3.

  • Martinez, A, Miller, MI, Quing, K, Unsworth, EJ, Ebina, M, Cutfitta, F (1995) Non-radioactive localization of nucleic acids by direct in situ PCR and in situ RT-PCR in paraffin-embedded sections. J. Histochem. Cytochem. 43: 739-747.

  • Stein, U, Walther, W, Wendt, J, Schfid, TA (1997) In situ RT-PCR using fluorescence-labeled primers. BioTechniques 23: 194-195.





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