4.5. cDNA synthesis from total RNA

Most downstream measurements of RNA traits rely on the complementary DNA (cDNA) generated by back-transcribing RNA using a commercially available reverse transcriptase such as ‘Superscript’ (Invitrogen). Reverse transcription is the most delicate step in RT-PCR. This step is very sensitive to inhibitors and contaminants in the sample (Ståhlberg et al., 2004b) such that the efficiency can vary between 0.5% and 95%. This efficiency is furthermore also strongly affected by both the absolute and relative amounts of target RNA in a sample, especially at very low levels of target (Ståhlberg et al., 2004a; 2004b), and by a variety of reaction conditions (Singh et al., 2000).

To minimize this variability, the RNA concentrations should be measured accurately by spectrophotometry (Qubit; Invitrogen), and a constant amount added to the cDNA reactions. If the RNA concentration is very low (<10 ng/µl), 100 ng neutral carrier tRNA can be added to the reaction prior to addition for cDNA synthesis stabilize reverse transcription and detection reliability. The final major parameter to optimise is the cDNA primer. Different target-specific cDNA primers (such as used in One-step RT-qPCR reactions), can have significantly different reverse transcription reaction efficiencies, which will affect the quantitative estimation of the targets in the sample (Bustin, 2000). A useful, practical approach is therefore to first prepare a fully representative cDNA ‘copy’ of the entire RNA population, using random ‘hexamer’ (6-nucleotide) primers. Such a complete cDNA population will have much less quantitative biases between different targets due to variable reverse transcriptase reaction efficiencies, allowing for more accurate quantitative comparison and normalisation between different targets. However, cDNA prepared with random primers can sometimes overestimate the original amount of target RNA (Zhang and Byrne, 1999). Another commonly used technique for sampling RNA pools is to use poly-dT primers targeting the polyadenylated stretch found at the 3’ end of most messenger RNAs and also on most of the honey bee viruses.

4.5.1 Reverse Transcription of RNA