The article by Stangegaard et al. entitled “Reverse transcription using random pentadecamer primers increases yield and quality of resulting cDNA” (BioTechniques 40:649–656; May 2006) did not provide sufficient details about the synthesis of the random primers used. The random primers were ordered at a standard oligonucleotide vendor (e.g., Sigma-Genosys, Haverhill, UK) as Nx, where x indicates the desired length of the primer and N indicates a wobble base (either A, C, G, or T). We have only tested standard desalted primers, which also are the cheapest. Upon receipt of primers, these were redissolved in diethylpyrocarbonate (DEPC)-treated water to reduce RNase-induced degradation of the RNA template.
R15 primers synthesized in 1 µmol scale and purified using desalting cost about $46 from Sigma-Genosys. This synthesis scale results in sufficient primers for more than 100 reverse transcription reactions using 3.35 nmol primer/reaction. Hence, R15 primers cost about $0.50/reaction. In contrast, the commercial random primer formulation costs almost $6/reaction ($300 for 50 reactions).
The study included only data from experiments using equimolar amounts of primer for both R15 and Inv hexamers (3.35 nmol primer/reaction, respectively). This corresponds to 15 µg R15 and 6 µg random hexamers. However, reverse transcription reactions primed with the same amount per weight of the respective random primers (i.e., 6 µg/reaction of either R15 or the commercial random primer) resulted in similar yields as reported in the article. This suggests that the amount of R15 primer could be decreased without affecting reaction yields significantly. However, as the R15 does not contribute significantly to the total reaction costs, we routinely used 3.35 nmol/reaction.
