The choice of primer for cDNA synthesis should reflect the downstream PCR requirements. Diagnostic real-time RT-PCR will require amplification of at least one control transcript and the specific test sequence. Therefore the use of specific reverse PCR primers to prime cDNA synthesis is not recommended as separate cDNA reactions would be required and subsequent analysis of other sequences would require further cDNA synthesis. However, if maximum sensitivity is required, cDNA synthesis using the downstream PCR primer will increase the yield of sequence specific PCR product.
Oligo d(T) priming utilizing the poly(A) tail present at the 3' end of most (but not all) mRNA molecules is used in many applications. It has the major advantage of priming cDNA synthesis almost exclusively from mRNA. However, for detection of fusion transcripts in leukemia, this method results in lower sensitivity, as many of the fusions occur towards the 5' end of the mRNA molecule and oligo d(T) primed cDNA is heavily biased towards 3' ends. It is also less tolerant of slightly degraded RNA templates.
Random priming using random hexamers is the method of choice for most applications in leukemia diagnosis and MRD monitoring. The cDNA produced is representative of the entire expressed RNA population; this does include the major component of cellular RNA the ribosomal RNA (rRNA), however this does not appear to compromise the sensitivity of realtime PCR. The Europe against Cancer (EAC) program designed and validated primer and probe combinations for the identification and monitoring of fusion transcripts in leukemia (Gabert et al., 2003). Optimal conditions for cDNA synthesis were also investigated and the recommended protocol is used in our laboratory.
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