Evaluation of digital PCR for absolute RNA quantification.
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.
Molecular and Cell Biology, LGC, Teddington, United Kingdom ; Cardiff School of BioSciences, The Sir Martin Evans Building, Cardiff, United Kingdom.
Rebecca
R
Sanders
Deborah J
DJ
Mason
Carole A
CA
Foy
Jim F
JF
Huggett
D000951:Antigens, Neoplasm / Q000235:genetics; D001859:Bone Neoplasms / Q000235:genetics; D006528:Carcinoma, Hepatocellular / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics*; D004720:Endonucleases / Q000235:genetics*; D018628:Gene Dosage; D005910:Glioma / Q000235:genetics; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics; D020781:Matrix Metalloproteinase 1 / Q000235:genetics; D012516:Osteosarcoma / Q000235:genetics; D012334:RNA, Neoplasm / Q000032:analysis / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D013056:Spectrophotometry, Ultraviolet; D014407:Tumor Cells, Cultured
D000951:Antigens, Neoplasm; D004268:DNA-Binding Proteins; D012334:RNA, Neoplasm; C440529:UBC antigen; C071447:ERCC1 protein, human; D004720:Endonucleases; C522360:MMP1 protein, human; D020781:Matrix Metalloproteinase 1