Quantitative assessment of the sensitivity of various commercial reverse transcriptases based on armored HIV RNA

Abstract

Background: The in-vitro reverse transcription of RNA to its complementary DNA, catalyzed by the enzyme reverse transcriptase, is the most fundamental step in the quantitative RNA detection in genomic studies. As such, this step should be as analytically sensitive, efficient and reproducible as possible, especially when dealing with degraded or low copy RNA samples. While there are many reverse transcriptases in the market, all claiming to be highly sensitive, there is need for a systematic independent comparison of their applicability in quantification of rare RNA transcripts or low copy RNA, such as those obtained from archival tissues.

Methodology/principal findings: We performed RT-qPCR to assess the sensitivity and reproducibility of 11 commercially available reverse transcriptases in cDNA synthesis from low copy number RNA levels. As target RNA, we used a serially known number of Armored HIV RNA molecules, and observed that 9 enzymes we tested were consistently sensitive to ∼1,000 copies, seven of which were sensitive to ∼100 copies, while only 5 were sensitive to ∼10 RNA template copies across all replicates tested. Despite their demonstrated sensitivity, these five best performing enzymes (Accuscript, HIV-RT, M-MLV, Superscript III and Thermoscript) showed considerable variation in their reproducibility as well as their overall amplification efficiency. Accuscript and Superscript III were the most sensitive and consistent within runs, with Accuscript and Superscript II ranking as the most reproducible enzymes between assays.

Conclusions/significance: We therefore recommend the use of Accuscript or Superscript III when dealing with low copy number RNA levels, and suggest purification of the RT reactions prior to downstream applications (eg qPCR) to augment detection. Although the results presented in this study were based on a viral RNA surrogate, and applied to nucleic acid lysates derived from archival formalin-fixed paraffin embedded tissue, their relative performance on RNA obtained from other tissue types may vary, and needs future evaluation.

Figure 1. Average quantification cycles (and their standard deviations, represented as error bars) from 10, 100, 1000 copies of Armored HIV RNA assayed using all 11 reverse transcriptases compared in this study.

The coloring corresponds to the copies of Armored RNA in each reaction. Only Cq points above 25 cycles are shown to emasize the differences among the enzymes assessed.

Figure 2. Standard curves for the five most reproducible reverse transcriptases that amplified all Armored HIV RNA replicates tested in this study.

The standard curves (solid lines) and their associated 95% confidence intervals (dashed lines) were generated by the Mxp-MX3000P software by plotting quantification cycles (Cq) against RNA molecules (10–1,000 copies) amplified in duplicates. Only Cq points above 25 are shown in the graphs to emphasize phthe differences among the enzymes assessed. Correlation coefficients (Rsq) and amplification efficiencies (Eff.) as well as the linear equation of each curve are shown. The tightness of the 95% confidence limit shows quantification accuracy given an input RNA template amount.

Figure 3. Comparison of amplification success measured by quantification cycle (Cq) of the straight cDNA with post-RT processed cDNA.

Post-RT processing was by means of dilutions, phenol-chloroform-isoamylalcohol-microcon and MiniElute purification of ACC generated RT products, the latter two in tripcates. A) Typical amplifications profile of a DNA standard (green), compared to ones spiked with RT unpurified (straight; in red) and PCI-Microcon YM-30 cleaned products (blue). The shift in Cq when the amplification reaction included straight RT products demonstrates the inhibitory effects of RTs. B), comparison of the strategies used to reduce inhibitory effects of RT, with blue bars showing dilutions (as percentage reduction of straight template) and increasing amounts of post-RT cleaned products (depicted as purple stripes, superimposed on yellow bars). Only Cq points above 25 were shown in the graphs to emphasize the differences revealed. Straight/Dilution = Straight Armored RNA templates range from 0.25–40%, and PCI-Microcon = phenol-chloroform microcon cleaned Armored RNA products.