In the RT-PCR method, RNA is initially reverse-transcribed to single-stranded cDNA and then the desired target cDNA species is amplified using specific primers. Fewer than 10 copies of target RNA are required for this procedure, and it has even been successful when the RNA was isolated from a single cell. In this protocol, the level of mRNA is quantified by RT-PCR and southern blot analysis.
4. Quantitate the signal on X-ray film by densitometer.
Alternatively, use a Phosphorimager (Molecular Dynamics) that is more sensitive and faster than conventional densitometry. This technique allows the capture of radioactive energy from the probe bound to the blot by exposure to a Phosphor storage screen. This signal is then transferred via the Phosphorimager and imagequant software to the computer screen, where the background signal and specific signal can be quantitated.
A negative control should be included during RT-PCR amplification and blotting. At the start of the Reverse Transcription reaction, a tube with no added RNA should be included. Instead of RNA, add an equivalent volume of water or RT buffer. This sample should be processed with the others and, following the RT reaction and PCR reaction, an aliquot from the PCR product should be southern blotted as a control for any contaminating DNA or primers. Ideally, there should be no signal in the lane for the negative control. If there is competing genomic contamination represented as a second band, the PCR primers should be redesigned.
A positive control for the southern blot should be included. RNA from a source known to express the gene of interest at levels comparable to the test samples is ideal. This control will allow determination of the specificity of the PCR. The control RNA sample should first be reverse-transcribed and then amplified to plateau level (30-40 cycles) and run on an agarose gel with ethidium bromide and size markers to determine if the expected size product is observed. Other contaminating bands may also be observed, but ideally there should only be one band. The signal from the experimental group should match the positive control in relative position on the blot.
Lack of signal from the positive control and the other samples indicates a problem with the probe or the blotting procedure. If lanes other than the positive control are labeled, it probably indicates an error in the PCR for the positive control. If the positive control hybridizes very weakly, it probably indicates a problem with denaturing, neutralizing, or saturating the gel; failure to transfer or to cross-link the blot properly may also be a problem.
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