Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data.
Quantification of mRNAs using real-time polymerase chain reaction (PCR) by monitoring the product formation with the fluorescent dye SYBR Green I is being extensively used in neurosciences, developmental biology, and medical diagnostics. Most PCR data analysis procedures assume that the PCR efficiency for the amplicon of interest is constant or even, in the case of the comparative C(t) method, equal to 2. The latter method already leads to a 4-fold error when the PCR efficiencies vary over just a 0.04 range. PCR efficiencies of amplicons are usually calculated from standard curves based on either known RNA inputs or on dilution series of a reference cDNA sample. In this paper we show that the first approach can lead to PCR efficiencies that vary over a 0.2 range, whereas the second approach may be off by 0.26. Therefore, we propose linear regression on the Log(fluorescence) per cycle number data as an assumption-free method to calculate starting concentrations of mRNAs and PCR efficiencies for each sample. A computer program to perform this calculation is available on request (e-mail: bioinfo@amc.uva.nl; subject: LinRegPCR).
Ramakers
Christian
C
Department of Anatomy and Embryology K2-283, Experimental and Molecular Cardiology Group, Academic Medical Centre, University of Amsterdam, Meibergdreef 15, 1105 AZ, Amsterdam, The Netherlands. c.r.ramakers@amc.uva.nl
Ruijter
Jan M
JM
Deprez
Ronald H Lekanne
RH
Moorman
Antoon F M
AF
D052160:Benzothiazoles; D002138:Calibration; D018076:DNA, Complementary / Q000032:analysis; D003959:Diamines; D005456:Fluorescent Dyes; D016014:Linear Models; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction / Q000592:standards* / Q000706:statistics & numerical data; D011804:Quinolines; D012333:RNA, Messenger / Q000032:analysis; D013997:Time Factors
D052160:Benzothiazoles; D018076:DNA, Complementary; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; D012333:RNA, Messenger; C098022:SYBR Green I