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Properties of the reverse transcription reaction in mRNA quantification.

Journal article
Authors Anders Ståhlberg
Joakim Håkansson
Xiaojie Xian
Henrik Semb
Mikael Kubista
Published in Clinical chemistry
Volume 50
Issue 3
Pages 509-15
ISSN 0009-9147
Publication year 2004
Published at Institute of Medical Biochemistry
Pages 509-15
Language en
Keywords Animals, Cell Line, Tumor, Gene Expression Profiling, Glucose Transporter Type 2, Glyceraldehyde-3-Phosphate Dehydrogenases, genetics, Insulin, genetics, Mice, Monosaccharide Transport Proteins, genetics, Pancreatic Neoplasms, RNA, Messenger, analysis, chemistry, RNA-Directed DNA Polymerase, chemistry, Reproducibility of Results, Reverse Transcriptase Polymerase Chain Reaction, methods, Sensitivity and Specificity, Tubulin, genetics
Subject categories Medical and Health Sciences


BACKGROUND: In most measurements of gene expression, mRNA is first reverse-transcribed into cDNA. We studied the reverse transcription reaction and its consequences for quantitative measurements of gene expression. METHODS: We used SYBR green I-based quantitative real-time PCR (QPCR) to measure the properties of reverse transcription reaction for the beta-tubulin, glyceraldehyde-3-phosphate dehydrogenase, Glut2, CaV1D, and insulin II genes, using random hexamers, oligo(dT), and gene-specific reverse transcription primers. RESULTS: Experimental variation in reverse transcription-QPCR (RT-QPCR) was mainly attributable to the reverse transcription step. Reverse transcription efficiency depended on priming strategy, and the dependence was different for the five genes studied. Reverse transcription yields also depended on total RNA concentration. CONCLUSIONS: RT-QPCR gene expression measurements are comparable only when the same priming strategy and reaction conditions are used in all experiments and the samples contain the same total amount of RNA. Experimental accuracy is improved by running samples in (at least) duplicate starting with the reverse transcription reaction.

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