Primer location in the sequence

Oligo dT and random primers are commonly used in reverse transcription (RT) to produce cDNA template for real-time PCR. Oligo dT primers anneal specifically to mRNA poly(A) tails, thus minimizing non-coding cDNA products. However, this priming strategy introduces 3' bias in cDNA synthesis because it is difficult to produce full-length cDNAs due to limited RT extension capability. If oligo dT primers are used in RT, the real-time PCR primers should be picked from the 3' region of a gene sequence to gain maximum assay sensitivity.

In contrast, random primers are often used in RT for full transcript coverage. Random primers can potentially bind to any site in a transcript sequence. Because all primers are extended toward the 5' end of the transcript, there will be a linear gradient of sequence representation, with highest cDNA abundance in the 5' regions. If random priming strategy is adopted, real-time PCR primers should be picked close to the 5' end of the target sequence for maximum sensitivity in real-time PCR.

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