Properties of the reverse transcription reaction in mRNA quantification.
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Properties of the reverse transcription reaction in mRNA quantification. / Ståhlberg, Anders; Håkansson, Joakim; Xian, Xiaojie; Semb, Tor Henrik; Kubista, Mikael.
In: Clinical Chemistry, Vol. 50, No. 3, 2004, p. 509-15.Research output: Contribution to journal › Journal article › Research › peer-review
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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
VL - 50
SP - 509
EP - 515
JO - Clinical Chemistry
JF - Clinical Chemistry
SN - 0009-9147
IS - 3
ER -
ID: 5240932