Reverse transcriptasepolymerase chain reaction RTPCR

An alternative to Northern blotting for detecting and estimating relative amounts of BCL-2 mRNA is RT-PCR. If performed in a semi-quantitative fashion, this technique can allow for monitoring of fluctuations in BCL-2 mRNA levels within cells and requires far fewer cells than Northern blotting experiments. We have used the RT-PCR approach, for example, to monitor the effects of antisense oligonucleotides targeted against the human BCL-2 mRNA, which induce an RNAse-H-like degradation of mRNAs, as well as for assessing the effects of p53 on bcl-2 mRNA levels in murine leukaemia cells (31-33).

Total RNA can be prepared by either the guanidinium isothiocyanate procedure described above (27) or using a commercially available, streamlined version of the method involving the TRIzolâ„¢ reagent (BRL/Gibco, Inc.). In some cases, it may be advisable to check 1 (xg of the RNA in a minigel (formaldehyde-containing) to assess integrity of the 28S and 18S rRNA bands, assess whether contaminating DNA is present, and verify the quantification results (i.e. the starting amount is approximately the same for all samples).

The procedure described below involves use of a recombinant version of Moloney Virus-derived RTase, SuperScriptâ„¢ (BRL/Gibco, Inc.) for the cDNA synthesis and Taq DNA polymerase (Perkin-Elmer, Inc.) for the PCR, but other RTases and heat-stable DNA polymerases may also be suitable.

It is important to also perform RT-PCR for the same sample of RNA using a control set of primers. For mouse, we typically target ^-microglobulin mRNA, whereas for human GADPH is used. The primer sequences for amplification of these control mRNAs are also provided. Of course, a variety of other control mRNAs could be equally, or even more, appropriate, depending on the particular cells of interest. The value of the control RT-PCR is that it permits the bcl-2 results to be normalized relative to a control that should (in most circumstances) not vary.

The procedures described here are designed specifically for detection of bcl-2 RNAs but can be adapted through use of alternative amplification primers for any of the Bcl-2 family proteins.

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