Abstract
Gene expression measurements detailing mRNA quantities are widely employed in molecular biology and are increasingly important in diagnostic fields. Reverse transcription (RT), necessary for generating complementary DNA, can be both inefficient and imprecise, but remains a quintessential RNA analysis tool using qPCR. This study developed a Transcriptomic Calibration Material and assessed the RT reaction using digital (d)PCR for RNA measurement. While many studies characterise dPCR capabilities for DNA quantification, less work has been performed investigating similar parameters using RT-dPCR for RNA analysis. RT-dPCR measurement using three, one-step RT-qPCR kits was evaluated using single and multiplex formats when measuring endogenous and synthetic RNAs. The best performing kit was compared to UV quantification and sensitivity and technical reproducibility investigated. Our results demonstrate assay and kit dependent RT-dPCR measurements differed significantly compared to UV quantification. Different values were reported by different kits for each target, despite evaluation of identical samples using the same instrument. RT-dPCR did not display the strong inter-assay agreement previously described when analysing DNA. This study demonstrates that, as with DNA measurement, RT-dPCR is capable of accurate quantification of low copy RNA targets, but the results are both kit and target dependent supporting the need for calibration controls.
Highlights
Measuring RNA by reverse transcription real-time quantitative PCR (RT-qPCR) is an established approach for investigating gene expression and viral diagnostics
One-Step Reverse transcription (RT)-qPCR Kit Comparison by dPCR Three commercially available kits were compared for quantitative performance by RT-dPCR
This study has shown that dPCR is capable of making precise measurements of synthetic and endogenous RNA molecules in a complex RNA background
Summary
Measuring RNA by reverse transcription real-time quantitative PCR (RT-qPCR) is an established approach for investigating gene expression and viral diagnostics. Many of the issues associated with differing RTase efficiencies may be sidestepped by taking advantage of the linear nature of RT and performing relative quantification, with the results expressed as fold changes, or by comparing to a standard curve that is affected by the limitations of the RT. Digital (d)PCR is continuing to gain recognition in the field as an extremely precise and reproducible methodology offering the potential for accurate, robust and highly sensitive measurement without the need for a standard curve [2]. DPCR expands the long established premise of molecular quantification by qPCR through facilitating measurement of individual target molecules. DPCR may offer the potential to maximise the accuracy, sensitivity and reproducibility of RNA measurements, for capabilities such as diagnostic mRNA profiling, biomarker analysis and monitoring of viral load The need for a calibration curve to assign a value is argued to be unnecessary [4,5,7,11,12,13], and this fact has quickly led to the notion that dPCR is calibration free [2]. dPCR may offer the potential to maximise the accuracy, sensitivity and reproducibility of RNA measurements, for capabilities such as diagnostic mRNA profiling, biomarker analysis and monitoring of viral load
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