Gene

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Gene-specific RT-PCR

This method was successful in our lab using prostate tissue and for our specific objectives. Investigators must be aware that they will need to tailor the following protocol for their own research objectives and tissue under study.

To generate first strand cDNA from mRNA, three priming methods are routinely used, depending on the quality of the starting RNA:

Oligo (dT)-based priming is preferred when the RNA is of high quality, since first strand polymerization should be continuous from the 3" end of the transcript to the desired sequence regardless of the length of the 3" UTR. Random hexamer priming is recommended for fragmented mRNA (lengths <500 bases) where there may be discontinuity between the desired sequence and the poly A tail. A primer that is specific to the gene of interest is best when amplifying low abundance transcripts.

The NCI Prostate Group has found that the most efficient method for first strand cDNA synthesis of mRNA from microdissected samples, which may contain fragmented mRNA, uses random hexamers, as they circumvent the possibility of disruptions in oligo (dT)-based first strand cDNA polymerization.

A: Reverse Transcription

1. Reagents

1. DEPC-dH2O (Research Genetics) RNase inhibitor, 20 U/µl (Perkin Elmer) dNTP, 250 µM (GenHunter) First strand buffer (Life Technologies) Random hexamer primers, 50 mM (Perkin Elmer) Superscript II (SSII) reverse transcriptase (Life Technologies)

2. Method

The protocol below may be used for amplifying individual transcripts from RNA recovered from microdissected Cell populations as described in RNA-based Studies of Microdissected Tissues .

TIP: The number of cells needed to perform gene-specific RT-PCR from microdissected samples is highly dependent on the quality of the tissue sample and the abundance level of the transcript(s) of interest. One thousand cells is recommended as a good starting point for gene-specific RT-PCR studies. However, reliable amplification can frequently be achieved using substantially fewer cells.

 

<center> <p> Control Type(+) RT(-) RTPositiveHigh-quality RNAHigh-quality RNANegativeDEPC-dH2ODEPC-dH2O</p> </center>

 

<center> <p>  </p> <p> <strong>(+) RT master reaction mixture/tube</strong></p> 1.0 µlDEPC-dH2O2.0 µlFirst strand RT buffer1.0 µldNTP0.5 µlRandom hexamer primers Total volume = 4.5 µl</center> <center> <p>  </p> <p> <strong>(-) RT master reaction mixture/tube</strong></p> 1.5 µlDEPC-dH2O2.0 µlFirst strand RT buffer1.0 µldNTP0.5 µlRandom hexamer primers Total volume = 5.0 µl</center> Nature

To RNA pellet, add 10 µl DEPC-H2O and 1 µl RNase inhibitor. Resuspend the RNA pellet with gentle tapping. Quick spin. Aliquot 5 µl into 2 sterile tubes for (+) and (-) RT reactions. For each batch of samples, prepare additional control tubes as follows, using either high-quality RNA or DEPC-dH2O in place of the 5 µl sample RNA: Prepare sufficient volume of the following +/-RT master reaction mixtures for all reaction tubes. Aliquot either 4.5 µl or 5.0 µl of the relevant master mix to the (+) and (-) RT tubes. Incubate at 65°C for 5 minutes, then at 25°C for 10 minutes. Add 0.5 µl SSII to all (+) RT tubes only. Incubate all tubes at 25°C for 10 minutes, then at 37°C for 40 minutes. Incubate at 95°C for 5 minutes to de the SSII. Quick spin.