Conditions de fiabilité des mesures d expression d génique g par RT- en cancérologie clinique J.D. Combes 1,2, G. Grelier 3, M. Laversanne 2, N. Voirin 1, S. Chabaud 1, R. Ecochard 2, C. Moyret-Lalle 3 & C. Lasset 1 1 Département de santé publique, Centre Léon Bérard, Lyon 2 Service de biostatistiques des Hospices Civils de Lyon, CNRS UMR 5580 3 INSERM U590, Centre Léon Bérard, Lyon
PCR,,, RT-... Polymerase Chain Reaction Real-Time quantitative PCR Reverse-Transcription ranscription Real-Time quantitative PCR Transcription reverse (ADNc) 2/10
RT- en cancérologie Cancer T47D Culture cellulaire Extraction RT- Transcription ARN ADNc Expression APC11 + Non Cancer MB45 9 Culture cellulaire Extraction RT- Transcription ARN ADNc Expression APC11-3/10
Plan expérimental T47D Culture cellulaire Extraction ARN Transcription ADNc MB45 9 4/10
BT BT-20 20 BT BT-474 474 Cal51 Cal51 Cama1 Cama1 HCC HCC-1937 1937 HS HS-57BT 57BT MCF MCF-7 MDA MDA-MB157 MB157 MDA MDA-MB231 MB231 MDA MDA-MB436 MB436 MDA MDA-MB453 MB453 MDA MDA-MB361 MB361 MDA MDA-MB459 MB459 MDA MDA-MB549 MB549 SK SK-Br3 Br3 T47D T47D UACC UACC-612 612 ZR ZR-75.1 75.1 Plan Plan exp expérimental rimental 5/10
Estimation de l erreur l de mesure Lignées cellulaires (18) 18 3 1 2 Culture cellulaire Extraction ARN Transcription ADNc Source de variabilité Inter lignée Culture Extraction ARN RT ET 6/10
Estimation de l erreur l de mesure Lignées cellulaires (18) 18 3 1 2 Culture cellulaire Extraction ARN Transcription ADNc Source de variabilité Inter lignée Culture Extraction ARN RT ET 1.7 1.3 1.2 2.0 0.26 7/10
Conditions de fiabilité 8/10
Conclusion Fiabilité des mesures d expression d génique g Murphy J, Bustin SA. Reliability Tichopad of A, real-time Kitchen R, reverse-transcription Riedmaier I, Becker C, PCR Ståhlberg in clinical A, Kubista diagnostics: M. Design and gold standard or substandard Étapes optimization pré-pcr of reverse-transcription Pfaffl MW. quantitative PCR experiments. Expert Rev Clin Mol Chem. Diagn. 2009 2009 The Oct;55(10):1816-23. ongoing Mar;9(2):187-97. evolution Review. PubMed of. PubMed PMID: 19643838. PMID: 19298142. Methods. 2010 Apr;50(4):215-6. PubMed PMID: 20215019. MIQE However, We recommend Guidelines this technology the (Minimum use of is sample still Information troubled replicates by Most advances were implemented in PCR method for itself but preferentially Publication significant of Quantitative technical to any other deficiencies. replicates Real-Time pre-pcr steps like sampling, nucleic acid stabilisation PCR) and RT when working Hence its with often-improper solid tissue, cell use culture, as a clinical and single tool cells, are still highly variable and and we has recommend public health the use implications, of RT replicates introduce lots of error in the quantification procedure. most recently demonstrated when working through with blood. its association with the measles, mumps and rubella vaccine/autism controversy. 9/10
Remerciements INSERM U590 - CLB Gaël Grelier Caroline Moyret-Lalle Alain Puisieux Références Dépt de Santé Publique - CLB Sylvie Chabaud Christine Lasset David Perol Nicolas Voirin Biostatistiques HCL René Ecochard Jean Iwaz Matthieu Laversanne Pascal Roy Fabien Subtil 1: Combes JD, Grelier G, Laversanne M, Voirin N, Chabaud S, Ecochard R, Lasset C, Moyret-Lalle C. Contribution of cell culture, RNA extraction, and reverse transcription to the measurement error in quantitative reverse transcription polymerase chain reaction-based gene expression quantification. Anal Biochem. 2009 Oct 1;393(1):29-35. Epub 2009 Jun 13. PubMed PMID: 19527676. 2: Murphy J, Bustin SA. Reliability of real-time reverse-transcription PCR in clinical diagnostics: gold standard or substandard Expert Rev Mol Diagn. 2009 Mar;9(2):187-97. Review. PubMed PMID: 19298142. 3: Pfaffl MW. The ongoing evolution of. Methods. 2010 Apr;50(4):215-6.PubMed PMID: 20215019. 4: Tichopad A, Kitchen R, Riedmaier I, Becker C, Ståhlberg A, Kubista M. Design and optimization of reversetranscription quantitative PCR experiments. Clin Chem. 2009 Oct;55(10):1816-23. Epub 2009 Jul 30. PubMed PMID: 19643838. 10/10
T47D BT_20 BT_474 HCC_1937 Cama_1 ZR75.1 MDA_MB157 MDA_MB231 MCF_7 UACC812 Cal_51 SKBr3 MDA_MB361 MDA_MB453 Données 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 Gene expression value + + + + + + + + + + + + + + + + + + MDA_MB549 MDA_MB436 HS_578T MDA_MB459
Estimation de l erreur l de mesure Erreur Erreur de Variabilité de mesure mesure due à due àl extraction à la interlignées la Reverse culture Transcription de l ARN cellulaires
Conditions de fiabilité