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 efficien...

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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
Online Access	Get full text
ISSN	0304-3940 1872-7972
DOI	10.1016/S0304-3940(02)01423-4

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Abstract	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).
AbstractList	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).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). 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). 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).
Author	Ramakers, Christian Moorman, Antoon F.M Ruijter, Jan M Deprez, Ronald H.Lekanne
Author_xml	– sequence: 1 givenname: Christian surname: Ramakers fullname: Ramakers, Christian email: c.r.ramakers@amc.uva.nl organization: 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 – sequence: 2 givenname: Jan M surname: Ruijter fullname: Ruijter, Jan M email: j.m.ruijter@amc.uva.nl organization: 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 – sequence: 3 givenname: Ronald H.Lekanne surname: Deprez fullname: Deprez, Ronald H.Lekanne organization: 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 – sequence: 4 givenname: Antoon F.M surname: Moorman fullname: Moorman, Antoon F.M organization: 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
BackLink	http://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=14535440$$DView record in Pascal Francis https://www.ncbi.nlm.nih.gov/pubmed/12618301$$D View this record in MEDLINE/PubMed
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PublicationDate_xml	– month: 03 year: 2003 text: 2003-03-13 day: 13
PublicationDecade	2000
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PublicationPlace_xml	– name: Shannon – name: Ireland
PublicationTitle	Neuroscience letters
PublicationTitleAlternate	Neurosci Lett
PublicationYear	2003
Publisher	Elsevier Ireland Ltd Elsevier
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SubjectTerms	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
Title	Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data
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