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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Bibliographic Details
Published in:PloS one 2013-09, Vol.8 (9), p.e75296-e75296
Main Authors: Sanders, Rebecca, Mason, Deborah J, Foy, Carole A, Huggett, Jim F
Format: Article
Language:English
Subjects:
Analysis
Antigens, Neoplasm - genetics
Biology
Bone Neoplasms - genetics
Calibration
Carcinoma, Hepatocellular - genetics
Complementary DNA
Deoxyribonucleic acid
Diagnostic systems
DNA
DNA-Binding Proteins - genetics
Endonucleases - genetics
Enzymes
Evaluation
Gene Dosage
Gene expression
Genetic testing
Glioma - genetics
Humans
Leukemia
Liver Neoplasms - genetics
Matrix Metalloproteinase 1 - genetics
Messenger RNA
Molecular biology
Multiplexing
Osteosarcoma - genetics
Polymerase chain reaction
Process controls
Real-Time Polymerase Chain Reaction - methods
Reproducibility
Reverse transcription
Ribonucleic acid
RNA
RNA, Neoplasm - analysis
RNA, Neoplasm - genetics
Spectrophotometry, Ultraviolet
Studies
Transcription (Genetics)
Tumor Cells, Cultured
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Description
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.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0075296