|
|
|
| To normalize the using of quantitative real-time reverse transcription PCR |
|
|
|
Abstract Quantitative real-time reverse transcription-PCR (RT-qPCR ) is an efficient tool to measure absolute transcript abundance and provides valuable quantitative information on gene expression of biologic samples from different sources. Thousands of research laboratories worldwide have embraced RT-qPCR as a frequently used method for measuring genes expression in transcript levels because of its relatively low cost, high precision, and high sensitivity, as well as flexibility and scalability. However, despite its popularity, more and more researchers begin to realize that the accuracy of RT-qPCR gene expression analysis depends largely on a proper normalization. However, the simplicity of the technology itself makes it vulnerable for abuse in experiments in which the operator does not perform the required quality control throughout the entire procedure. Here, we review entire RT-qPCR workflow and indicated point by point where and how critical issues can be resolved, such as experiment design, sample and assay quality control, selection of proper reference genes for normalization, data analysis and reporting guidelines. Following the advice in this paper, any user should be able to do (more) successful gene expression profiling using the RT-qPCR technology.
|
|
Received: 01 June 2010
Published: 19 October 2010
|
|
|
| 参考文献
[1].Nolan T, Hands RE, Bustin SA, et al. Quantification of mRNA using real-time PCR. Nat. Protoc., 2006,1(3):1559-1582.
[2].Vandesompele J, De Preter K, Pattyn F, et al .Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes, Genome Biol. 2002, 3(7):RESEARCH0034. Epub 2002 Jun 18.
[3].Derveaux S, Vandesompele J, Hellemans J. How to do successful gene expression analysis using real-time PCR.Methods, 2010, 50(4):227-230.
[4].Bernard PS, Wittwer CT. Real-time PCR technology for cancer diagnostics. Clin Chem, 2002, 48(8):1178-1185.
[5].Bustin SA, Mueller R.Real-time reverse transcription PCR (RT-qPCR) and its potential use in clinical diagnosis. Clin Sci (Lond), 2005, 109(4):365-379.
[6].Bustin SA, Nolan T. Pitfalls of quantitative realtime reverse-transcription polymerase chain reaction. J Biomol Tech, 2004, 15(3):155-166.
[7].Hellemans J, Mortier G, De Paepe A, et al. qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biol, 2007, 8(2):R19.
[8]. Fleige S, Pfaffl MW. RNA integrity and the effect on the real-time RT-qPCR performance. Mol Aspects Med. 2006，27(2-3):126-139.
[9]. Huggett JF, Novak T, Garson JA, et al. Differential susceptibility of PCR reactions to inhibitors: an important and unrecognised phenomenon. BMC Res Notes, 2008,28 (1):70.
[10].Nolan T, Hands RE, Ogunkolade W, et al. SPUD: a quantitative PCR assay for the detection of inhibitors in nucleic acid preparations. Anal Biochem., 2006, 351(2): 308-310.
[11].Hoebeeck J, van der Luijt R, Poppe B, et al. Rapid detection of VHL exon deletions using real-time quantitative PCR. Lab Invest，2005，85(1):24-33.
[12]. Pattyn F, Robbrecht P, De Paepe A, et al. RT Primer DB: the real-time PCR primer and probe database, major update 2006. Nucleic Acids Res, 2006, 34:D684–688.
[13].Andersen CL, Jensen JL, Orntoft TF. 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 datasets. Cancer Res, 2004, 64(15):5245-5250.
[14].Vandesompele J, De Preter K, Pattyn F, et al . GeNorm software manual, update 6 2004. Available from: .
[15].NormFinder Software. Available from: .
[16].Segers-Nolten GM, Wyman C, Wijgers N, et al. Scanning confocal fluorescence microscopy for single molecule analysis of nucleotide excision repair complexes. Nucleic Acids Res，2002，30(21):4720-4727.
[17].Karlen Y, McNair A, Perseguers S, et al. Statistical significance of quantitative PCR. BMC Bioinformatics, 2007, 20(8):131.
[18].Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc, 2008, 3(6):1101-1108.
[19].Hellemans J, Preobrazhenska O, Willaert A, et al. Loss-of-function mutations in LEMD3 result in osteopoikilosis, Buschke-Ollendorff syndrome and melorheostosis. Nat Genet, 2004, 36(11):1213-1218.
[20].Willems E, Leyns L, Vandesompele J. Standardization of real-time PCR gene expression data from independent biological replicates. Anal Biochem, 2008, 379(1):127-129.
[21].Pfaffl MW, Horgan GW, Dempfle L. Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res, 2002, 30(9):e36.
[22].Lefever S, Hellemans J, Pattyn F, et al. RDML: structured language and reporting guidelines for real-time quantitative PCR data. Nucleic Acids Res, 2009, 37(7):2065-2069.
[23].Bustin SA, Benes V, Garson JA, et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem, 2009, 55(4):611-622.
|
|
Viewed |
|
|
|
Full text
|
|
|
|
|
Abstract
|
|
|
|
|
Cited |
|
|
|
|
| |
Shared |
|
|
|
|
| |
Discussed |
|
|
|
|
