Properties of the reverse transcription reaction in mRNA quantification.
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.
Ståhlberg
Anders
A
Department of Chemistry and Bioscience, Chalmers University of Technology, Gothenburg, Sweden.
Håkansson
Joakim
J
Xian
Xiaojie
X
Semb
Henrik
H
Kubista
Mikael
M
D000818:Animals; D045744:Cell Line, Tumor; D020869:Gene Expression Profiling; D051273:Glucose Transporter Type 2; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D007328:Insulin / Q000235:genetics; D051379:Mice; D009004:Monosaccharide Transport Proteins / Q000235:genetics; D010190:Pancreatic Neoplasms; D012333:RNA, Messenger / Q000032:analysis* / Q000737:chemistry; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014404:Tubulin / Q000235:genetics
D051273:Glucose Transporter Type 2; D007328:Insulin; D009004:Monosaccharide Transport Proteins; D012333:RNA, Messenger; D014404:Tubulin; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D012194:RNA-Directed DNA Polymerase