Standardization of real-time PCR gene expression data from independent biological replicates.
Gene expression analysis by quantitative reverse transcription PCR (qRT-PCR) allows accurate quantifications of messenger RNA (mRNA) levels over different samples. Corrective methods for different steps in the qRT-PCR reaction have been reported; however, statistical analysis and presentation of substantially variable biological repeats present problems and are often not meaningful, for example, in a biological system such as mouse embryonic stem cell differentiation. Based on a series of sequential corrections, including log transformation, mean centering, and autoscaling, we describe a robust and powerful standardization method that can be used on highly variable data sets to draw statistically reliable conclusions.
Willems
Erik
E
Del E. Webb Neuroscience, Aging, and Stem Cell Research Center, Burnham Institute for Medical Research, La Jolla, CA 92037, USA. ewillems@burnham.org
Leyns
Luc
L
Vandesompele
Jo
J
D000818:Animals; D053595:Embryonic Stem Cells / Q000378:metabolism; D015870:Gene Expression / Q000235:genetics*; D051379:Mice; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012015:Reference Standards; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards
D012333:RNA, Messenger