Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data

• Published in: Neuroscience letters Vol. 339; no. 1; pp. 62 - 66
• Main Authors: Ramakers, Christian, Ruijter, Jan M, Deprez, Ronald H.Lekanne, Moorman, Antoon F.M
• Format: Journal Article
• Language: English
• Published: Shannon Elsevier Ireland Ltd 13.03.2003; Elsevier
• Subjects: Biological and medical sciences; Calibration; Comparative Ct method; DNA, Complementary - analysis; Fluorescent Dyes; Fundamental and applied biological sciences. Psychology; General aspects. Models. Methods; Linear Models; Linear regression; mRNA quantification; Organic Chemicals; PCR efficiency; Polymerase Chain Reaction - standards; Polymerase Chain Reaction - statistics & numerical data; Quantitative polymerase chain reaction (PCR); Real-time PCR; RNA, Messenger - analysis; Standard curve method; SYBR ® green I; Time Factors; Vertebrates: nervous system and sense organs; Quantitative polymerase chain reaction (PCR); Comparative C t method; SYBR ® green I; Real-time PCR; mRNA quantification; Linear regression; PCR efficiency; Standard curve method; Comparative C, method; SYBR® green I; Real time; Polymerase chain reaction; Messenger RNA; Technique; Quantitative analysis
• ISSN: 0304-3940; 1872-7972
• DOI: 10.1016/S0304-3940(02)01423-4

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).