Properties of the reverse transcription reaction in mRNA quantification
A Stahlberg, J Hakansson, X Xian, H Semb… - Clinical …, 2004 - academic.oup.com
Clinical chemistry, 2004•academic.oup.com
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 β-tubulin, glyceraldehyde-3-phosphate dehydrogenase, Glut2, CaV1D, and
insulin II genes, using random hexamers, oligo (dT), and gene-specific reverse transcription …
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 β-tubulin, glyceraldehyde-3-phosphate dehydrogenase, Glut2, CaV1D, and
insulin II genes, using random hexamers, oligo (dT), and gene-specific reverse transcription …
Abstract
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 β-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.
Oxford University Press