Correction of RT-qPCR Data for Genomic DNA-derived Signals with ValidPrime

Overview

Journal Nucleic Acids Res

Publisher Oxford University Press

Specialty Biochemistry

Date 2012 Jan 10

PMID 22228834

Citations 40

Authors

Henrik Laurell

Jason S Iacovoni

Anne Abot

David Svec

Jean-Jose Maoret

Jean-Francois Arnal

Mikael Kubista

Affiliations

Soon will be listed here.

Abstract

Genomic DNA (gDNA) contamination is an inherent problem during RNA purification that can lead to non-specific amplification and aberrant results in reverse transcription quantitative PCR (RT-qPCR). Currently, there is no alternative to RT(-) controls to evaluate the impact of the gDNA background on RT-PCR data. We propose a novel method (ValidPrime) that is more accurate than traditional RT(-) controls to test qPCR assays with respect to their sensitivity toward gDNA. ValidPrime measures the gDNA contribution using an optimized gDNA-specific ValidPrime assay (VPA) and gDNA reference sample(s). The VPA, targeting a non-transcribed locus, is used to measure the gDNA contents in RT(+) samples and the gDNA reference is used to normalize for GOI-specific differences in gDNA sensitivity. We demonstrate that the RNA-derived component of the signal can be accurately estimated and deduced from the total signal. ValidPrime corrects with high precision for both exogenous (spiked) and endogenous gDNA, contributing ∼60% of the total signal, whereas substantially reducing the number of required qPCR control reactions. In conclusion, ValidPrime offers a cost-efficient alternative to RT(-) controls and accurately corrects for signals derived from gDNA in RT-qPCR.

Citing Articles

R-Ras1 and R-Ras2 regulate mature oligodendrocyte subpopulations.

Alcover-Sanchez B, Garcia-Martin G, Paleo-Garcia V, Quintas A, Dopazo A, Gruart A Glia. 2024; 73(4):701-719.

PMID: 39558879 PMC: 11845848. DOI: 10.1002/glia.24643.

CleanUpRNAseq: An R/Bioconductor Package for Detecting and Correcting DNA Contamination in RNA-Seq Data.

Liu H, Hu K, OConnor K, Kelliher M, Zhu L BioTech (Basel). 2024; 13(3).

PMID: 39189209 PMC: 11348166. DOI: 10.3390/biotech13030030.

Focused cancer pathway analysis revealed unique therapeutic targets in retinoblastoma.

Balaji S, Rao A, Saraswathi K, Sethu Nagarajan R, Santhi R, Kim U Med Oncol. 2024; 41(7):168.

PMID: 38834895 DOI: 10.1007/s12032-024-02391-9.

Predicting sepsis using a combination of clinical information and molecular immune markers sampled in the ambulance.

Tuerxun K, Eklund D, Wallgren U, Dannenberg K, Repsilber D, Kruse R Sci Rep. 2023; 13(1):14917.

PMID: 37691028 PMC: 10493220. DOI: 10.1038/s41598-023-42081-6.

Gene Expression Quantification from Pathogenic Bacterial Biofilms by Quantitative PCR.

Franca A, Cerca N Methods Mol Biol. 2023; 2967:133-149.

PMID: 37608108 DOI: 10.1007/978-1-0716-3358-8_11.

References

Yun J, Heisler L, Hwang I, Wilkins O, Lau S, Hyrcza M . Genomic DNA functions as a universal external standard in quantitative real-time PCR. Nucleic Acids Res. 2006; 34(12):e85. PMC: 1524913. DOI: 10.1093/nar/gkl400. View

Liu Y, Zheng D, Balasubramanian S, Carriero N, Khurana E, Robilotto R . Comprehensive analysis of the pseudogenes of glycolytic enzymes in vertebrates: the anomalously high number of GAPDH pseudogenes highlights a recent burst of retrotrans-positional activity. BMC Genomics. 2009; 10:480. PMC: 2770531. DOI: 10.1186/1471-2164-10-480. View

Nordgard O, Kvaloy J, Farmen R, Heikkila R . Error propagation in relative real-time reverse transcription polymerase chain reaction quantification models: the balance between accuracy and precision. Anal Biochem. 2006; 356(2):182-93. DOI: 10.1016/j.ab.2006.06.020. View

Roy S, Gilbert W . The evolution of spliceosomal introns: patterns, puzzles and progress. Nat Rev Genet. 2006; 7(3):211-21. DOI: 10.1038/nrg1807. View

Peccoud J, Jacob C . Theoretical uncertainty of measurements using quantitative polymerase chain reaction. Biophys J. 1996; 71(1):101-8. PMC: 1233461. DOI: 10.1016/S0006-3495(96)79205-6. View

Nolan T, Hands R, Bustin S . Quantification of mRNA using real-time RT-PCR. Nat Protoc. 2007; 1(3):1559-82. DOI: 10.1038/nprot.2006.236. View

Spurgeon S, Jones R, Ramakrishnan R . High throughput gene expression measurement with real time PCR in a microfluidic dynamic array. PLoS One. 2008; 3(2):e1662. PMC: 2244704. DOI: 10.1371/journal.pone.0001662. View

Pfaffl M . A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 2001; 29(9):e45. PMC: 55695. DOI: 10.1093/nar/29.9.e45. View

Calippe B, Douin-Echinard V, Laffargue M, Laurell H, Rana-Poussine V, Pipy B . Chronic estradiol administration in vivo promotes the proinflammatory response of macrophages to TLR4 activation: involvement of the phosphatidylinositol 3-kinase pathway. J Immunol. 2008; 180(12):7980-8. DOI: 10.4049/jimmunol.180.12.7980. View

10.

Andersen C, Jensen J, Orntoft T . Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res. 2004; 64(15):5245-50. DOI: 10.1158/0008-5472.CAN-04-0496. View

11.

Bustin S, Benes V, Garson J, Hellemans J, Huggett J, Kubista M . The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 2009; 55(4):611-22. DOI: 10.1373/clinchem.2008.112797. View

12.

Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A . Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002; 3(7):RESEARCH0034. PMC: 126239. DOI: 10.1186/gb-2002-3-7-research0034. View

13.

Hellemans J, Mortier G, De Paepe A, Speleman F, Vandesompele J . qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biol. 2007; 8(2):R19. PMC: 1852402. DOI: 10.1186/gb-2007-8-2-r19. View

14.

Riant E, Waget A, Cogo H, Arnal J, Burcelin R, Gourdy P . Estrogens protect against high-fat diet-induced insulin resistance and glucose intolerance in mice. Endocrinology. 2009; 150(5):2109-17. DOI: 10.1210/en.2008-0971. View

15.

Hofstetter J, Zhang A, Mayeda A, Guscar T, Nurnberger Jr J, Lahiri D . Genomic DNA from mice: a comparison of recovery methods and tissue sources. Biochem Mol Med. 1998; 62(2):197-202. DOI: 10.1006/bmme.1997.2637. View

16.

Higuchi R, Fockler C, Dollinger G, Watson R . Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. Biotechnology (N Y). 1993; 11(9):1026-30. DOI: 10.1038/nbt0993-1026. View

17.

Livak K, Schmittgen T . Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2002; 25(4):402-8. DOI: 10.1006/meth.2001.1262. View

18.

Gibson U, Heid C, Williams P . A novel method for real time quantitative RT-PCR. Genome Res. 1996; 6(10):995-1001. DOI: 10.1101/gr.6.10.995. View

19.

Bengtsson M, Hemberg M, Rorsman P, Stahlberg A . Quantification of mRNA in single cells and modelling of RT-qPCR induced noise. BMC Mol Biol. 2008; 9:63. PMC: 2483285. DOI: 10.1186/1471-2199-9-63. View

20.

Bustin S . Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J Mol Endocrinol. 2002; 29(1):23-39. DOI: 10.1677/jme.0.0290023. View