Table 1.
Sample dilutions analysed. Derived by UV spectrophotometry (ERCCs only).
Figure 1.
Three different one-step RT-qPCR kits were compared in both uniplex and duplex formats, by dPCR. Two external targets, ERCC-25 and ERCC-99 were analysed. Error bars: 95% Confidence intervals. n = 3 replicate panels. Equivalent UV estimates: ERCC-25 1185 copies/panel, 95% CI 17.34. ERCC-99 1185 copies/panel, 95% CI 26.19.
Table 2.
Three one-step kit comparison with uniplex and duplex formats.
Figure 2.
dPCR versus UV quantification.
Six external targets (ERCC-13, −25, −42, −99, −113 and −171) were assessed by both one-step dPCR, utilising the Ambion one-step RT-qPCR kit, and UV measurement. Error bars: 95% Confidence intervals. n = 3 replicate dPCR experiments or UV measurements.
Figure 3.
dPCR sensitivity for RNA measurement.
Assessment of RT-dPCR quantification sensitivity, using independent dilutions and quantifying ERCC-25 and ERCC-99 external targets in a duplex format. n = 6 panels per dilution, plus two replicate experiments. UV data based on initial UV quantification of stock and predicted target levels following volumetric dilutions. (A) & (B) dPCR sensitivity. (B) Focus on lowest level target dilutions. Error bars: 95% Confidence intervals. (C) Precision of dPCR quantification compared to UV.
Figure 4.
Evaluation of Reverse Transcriptases.
Three different one-step RT-qPCR kits were compared in different duplex formats, by dPCR. Quantification for external (ERCC-25 and ERCC-99) and endogenous (MMP1 and UBC) targets was evaluated. ERCC-25 with ERCC-99 (duplex A), UBC with MMP1 (duplex B), and ERCC-25 with UBC (duplex C). In the key/tabulated values, the assay in brackets is the duplex partner for the assay whose positive partition values are being displayed. Error bars: 95% Confidence intervals. n = 3 replicate panels, plus two replicate experiments.