![]() ![]() We compared an oligo(dT)-primed reaction to a random-primed control reaction. All reactions used standard incubation times of 50–60 minutes. We tested the ability of each RT to reverse transcribe an 8.9kb adenomatosis polyposis coli (APC) transcript from human total RNA. Without efficient extension during the RT reaction, portions of the transcripts may be lost to further analysis. Reverse transcription of full-length mRNAs is critical for cloning or when the PCR target is at the 5´ end of a transcript that will be oligo(dT)-primed. Based on these results, we concluded there is no dramatic distinction in the utility of these RTs with regard to sensitivity of transcript detection in reverse transcription-quantitative PCR(3). For the lower-abundance trp53 and cdk9 transcripts, detection was lost for all RTs at 10 and 1,000 cells, respectively, regardless of which enzyme was used (Figure 1, Panels C and D). ![]() Only GoScript™ Reverse Transcriptase allowed linear detection of lamin A down to a single cell (Figure 1, Panel B). The following four transcripts were analyzed (listed from highest to lowest abundance): glyceraldehyde-3-phosphate dehydrogenase (GAPDH), lamin A, transformation-related protein 53 (trp53) and cyclin-dependent kinase 9 (cdk9).Īll RTs allowed detection down to a single cell of the high-abundance transcript GAPDH (Figure 1, Panel A). Two microliters of each cDNA synthesis reaction then was analyzed using TaqMan® real-time PCR (Applied Biosystems). (Note: The PCR conditions used also will influence detection sensitivity.) To compare RT sensitivities of transcript detection, we used each enzyme and oligo(dT) as the primer to reverse transcribe serial dilutions of mouse liver total RNA representing 1 to 10,000 cell equivalents (assuming 20pg of total RNA per cell). The ideal RT will reverse transcribe even the least abundant mRNAs within a pool, allowing single-cell transcript detection. Gene expression analysis is the most common use for RTs. We also compared SuperScript® III and GoScript® reverse transcriptases for their ability to transcribe a challenging GC-rich mRNA. We compared these five RTs for their utility in detecting trace transcripts by real-time PCR, ability to transcribe a long RNA and sensitivity to ethanol, a common contaminant in RNA preparations. We performed a head-to-head comparison of five popular trademarked RTs: GoScript™ ( Promega Cat.# A5003), SuperScript® II and SuperScript® III (Invitrogen Cat.# 1800, respectively), and Omniscript® and Sensiscript® (Qiagen Cat.# 205113, respectively) reverse transcriptases. Genetic engineering and development of proprietary RT-enhancing buffers have led to the commercial availability of new enzymes that offer superior performance over these naturally occurring RTs. The most characterized RTs used for molecular biology are the retroviral RTs: avian myeloblastosis virus (AMV) and Moloney murine leukemia virus (MMLV). The ideal reverse transcriptase is robust (highly active under a variety of conditions) and converts all primed RNA within a sample to cDNA, regardless of its abundance, length or secondary structure. RT activity is critical for many basic techniques including real-time and endpoint RT-PCR, labeled-probe generation for microarrays and cDNA cloning. In addition to the critical role these enzymes play in a variety of major human diseases (HIV, hepatitis and some cancers), RTs are a fundamental component of the molecular biologist’s toolbox. Reverse transcriptases (RTs), also known as RNA-directed DNA polymerases, were first identified in retroviruses(1) (2). These data support the use of GoScript™ Reverse Transcriptase as a high-quality reverse transcriptase for gene expression analysis. GoScript™ Reverse Transcriptase outperformed SuperScript® III. ![]() We further compared GoScript™ and SuperScript® III reverse transcriptases using a difficult transcript and the standard elevated reaction temperature recommended for SuperScript® III (50☌). The new Promega GoScript™ Reverse Transcriptase performed as well as or better than Invitrogen’s SuperScript® II and SuperScript® III and Qiagen’s Omniscript® and Sensiscript® reverse transcriptases in terms of sensitivity of transcript detection, transcript length and sensitivity to ethanol contamination. We performed a direct functional comparison of popular reverse transcriptases to determine which offers superior performance. The ideal reverse transcriptase will robustly reverse transcribe from the priming site to the 5´ end of RNAs. Accurate gene expression analysis depends on the reverse transcriptase used as well as other factors (e.g., RNA quality and detection method). ![]()
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