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 analysi...
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Published in | PloS one Vol. 8; no. 9; p. e75296 |
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Main Authors | , , , |
Format | Journal Article |
Language | English |
Published |
United States
Public Library of Science
20.09.2013
Public Library of Science (PLoS) |
Subjects | |
Online Access | Get full text |
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Summary: | 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. |
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Bibliography: | ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Undefined-1 ObjectType-Feature-3 content type line 23 Competing Interests: The affiliation of authors Rebecca Sanders, Carole Foy and Jim Huggett with the commercial company LGC does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials. Conceived and designed the experiments: RS DJM CAF JFH. Performed the experiments: RS. Analyzed the data: RS JFH. Wrote the paper: RS JFH. |
ISSN: | 1932-6203 1932-6203 |
DOI: | 10.1371/journal.pone.0075296 |