Gene expression analysis is the most common use for RTs. The ideal RT will reverse transcribe even the least abundant mRNAs within a pool, allowing single-cell transcript detection. (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). Two microliters of each cDNA synthesis reaction then was analyzed using TaqMan® real-time PCR (Applied Biosystems). 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).
All RTs allowed detection down to a single cell of the high-abundance transcript GAPDH (Figure 1, Panel A). Only GoScript™ Reverse Transcriptase allowed linear detection of lamin A down to a single cell (Figure 1, Panel B). 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). 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) .