AAT Bioquest

What are reverse transcription primers?

Posted May 9, 2020


A reverse transcription primer is a short DNA oligo that binds to its complementary sequences on the RNA template and initiates the reverse transcriptase reaction, serving as the starting point for synthesis of a new DNA strand (i.e. cDNA). Three types of reverse transcription primers are usually used in research: oligo(dT) primers, random primers, and gene-specific primers.

Oligo(dT) primers consist of 12-18 deoxythymidines that anneal to the poly(A) tails of eukaryotic mRNAs, which are optimal for full-length DNA cloning but not suitable for degraded RNA or RNA with significant secondary structure. Random primers are oligonucleotides (mostly hexamers) with random base sequences. They can potentially anneal to any RNA species, thus are usually used for reverse transcription of RNAs without poly(A) tails as well as degraded RNA and RNA with known secondary structure. Gene-specific primers are designed based on the known sequence of the target RNA, which are commonly used in one-step RT-PCR applications.

Additional resources

StrandBrite™ Green RNA Quantifying Reagent *200X DMSO Solution*

Ohan, N. W., & Heikkila, J. J. (1993). Reverse transcription-polymerase chain reaction: An overview of the technique and its applications. Biotechnology Advances, 11(1), 13–29. doi:10.1016/0734-9750(93)90408-f

Nam, D. K., Lee, S., Zhou, G., Cao, X., Wang, C., Clark, T., … Wang, S. M. (2002). Oligo(dT) primer generates a high frequency of truncated cDNAs through internal poly(A) priming during reverse transcription. Proceedings of the National Academy of Sciences, 99(9), 6152–6156. doi:10.1073/pnas.092140899