Properties of the reverse transcription reaction in mRNA quantification.

TY - JOUR

T1 - Properties of the reverse transcription reaction in mRNA quantification.

AU - Ståhlberg, Anders

AU - Håkansson, Joakim

AU - Xian, Xiaojie

AU - Semb, Tor Henrik

AU - Kubista, Mikael

N1 - Keywords: Animals; Cell Line, Tumor; Gene Expression Profiling; Glucose Transporter Type 2; Glyceraldehyde-3-Phosphate Dehydrogenases; Insulin; Mice; Monosaccharide Transport Proteins; Pancreatic Neoplasms; RNA, Messenger; RNA-Directed DNA Polymerase; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction; Sensitivity and Specificity; Tubulin

PY - 2004

Y1 - 2004

N2 - 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.

AB - 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.

U2 - 10.1373/clinchem.2003.026161

DO - 10.1373/clinchem.2003.026161

M3 - Journal article

C2 - 14726469

SN - 0009-9147

VL - 50

SP - 509

EP - 515

JO - Clinical Chemistry

JF - Clinical Chemistry

IS - 3

ER -