Reference Genes for Real-time PCR Quantification of MicroRNAs and Messenger RNAs in Rat Models of Hepatotoxicity

Overview

Journal PLoS One

Specialties General Medicine
Science

Date 2012 May 8

PMID 22563491

Citations 52

Authors

Maria N Lardizabal

Ana L Nocito

Stella M Daniele

Leonardo A Ornella

Javier F Palatnik

Luis M Veggi

Affiliations

Soon will be listed here.

Abstract

Hepatotoxicity is associated with major changes in liver gene expression induced by xenobiotic exposure. Understanding the underlying mechanisms is critical for its clinical diagnosis and treatment. MicroRNAs are key regulators of gene expression that control mRNA stability and translation, during normal development and pathology. The canonical technique to measure gene transcript levels is Real-Time qPCR, which has been successfully modified to determine the levels of microRNAs as well. However, in order to obtain accurate data in a multi-step method like RT-qPCR, the normalization with endogenous, stably expressed reference genes is mandatory. Since the expression stability of candidate reference genes varies greatly depending on experimental factors, the aim of our study was to identify a combination of genes for optimal normalization of microRNA and mRNA qPCR expression data in experimental models of acute hepatotoxicity. Rats were treated with four traditional hepatotoxins: acetaminophen, carbon tetrachloride, D-galactosamine and thioacetamide, and the liver expression levels of two groups of candidate reference genes, one for microRNA and the other for mRNA normalization, were determined by RT-qPCR in compliance with the MIQE guidelines. In the present study, we report that traditional reference genes such as U6 spliceosomal RNA, Beta Actin and Glyceraldehyde-3P-dehydrogenase altered their expression in response to classic hepatotoxins and therefore cannot be used as reference genes in hepatotoxicity studies. Stability rankings of candidate reference genes, considering only those that did not alter their expression, were determined using geNorm, NormFinder and BestKeeper software packages. The potential candidates whose measurements were stable were further tested in different combinations to find the optimal set of reference genes that accurately determine mRNA and miRNA levels. Finally, the combination of MicroRNA-16/5S Ribosomal RNA and Beta 2 Microglobulin/18S Ribosomal RNA were validated as optimal reference genes for microRNA and mRNA quantification, respectively, in rat models of acute hepatotoxicity.

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References

Xing W, Deng M, Zhang J, Huang H, Dirsch O, Dahmen U . Quantitative evaluation and selection of reference genes in a rat model of extended liver resection. J Biomol Tech. 2009; 20(2):109-15. PMC: 2685606. View

Mestdagh P, Van Vlierberghe P, De Weer A, Muth D, Westermann F, Speleman F . A novel and universal method for microRNA RT-qPCR data normalization. Genome Biol. 2009; 10(6):R64. PMC: 2718498. DOI: 10.1186/gb-2009-10-6-r64. View

Li J, Guo H, Shi Z, Tu C . In vitro inhibition of CSFV replication by retroviral vector-mediated RNA interference. J Virol Methods. 2010; 169(2):316-21. PMC: 7112837. DOI: 10.1016/j.jviromet.2010.07.036. 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

Wang K, Zhang S, Marzolf B, Troisch P, Brightman A, Hu Z . Circulating microRNAs, potential biomarkers for drug-induced liver injury. Proc Natl Acad Sci U S A. 2009; 106(11):4402-7. PMC: 2657429. DOI: 10.1073/pnas.0813371106. View

Wang G, Xu C . Reference gene selection for real-time RT-PCR in eight kinds of rat regenerating hepatic cells. Mol Biotechnol. 2010; 46(1):49-57. DOI: 10.1007/s12033-010-9274-5. View

Nishimura M, Yamaguchi M, Yamauchi A, Ueda N, Naito S . Role of soybean oil fat emulsion in the prevention of hepatic xenobiotic transporter mRNA up- and down-regulation induced by overdose of fat-free total parenteral nutrition in infant rats. Drug Metab Pharmacokinet. 2005; 20(1):46-54. DOI: 10.2133/dmpk.20.46. View

Pfaffl M, Tichopad A, Prgomet C, Neuvians T . Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper--Excel-based tool using pair-wise correlations. Biotechnol Lett. 2004; 26(6):509-15. DOI: 10.1023/b:bile.0000019559.84305.47. View

Fox B, Devonshire A, Schutte M, Foy C, Minguez J, Przyborski S . Validation of reference gene stability for APAP hepatotoxicity studies in different in vitro systems and identification of novel potential toxicity biomarkers. Toxicol In Vitro. 2010; 24(7):1962-70. DOI: 10.1016/j.tiv.2010.08.007. View

10.

Chang K, Mestdagh P, Vandesompele J, Kerin M, Miller N . MicroRNA expression profiling to identify and validate reference genes for relative quantification in colorectal cancer. BMC Cancer. 2010; 10:173. PMC: 2873395. DOI: 10.1186/1471-2407-10-173. View

11.

Bargaje R, Hariharan M, Scaria V, Pillai B . Consensus miRNA expression profiles derived from interplatform normalization of microarray data. RNA. 2009; 16(1):16-25. PMC: 2802026. DOI: 10.1261/rna.1688110. View

12.

Peltier H, Latham G . Normalization of microRNA expression levels in quantitative RT-PCR assays: identification of suitable reference RNA targets in normal and cancerous human solid tissues. RNA. 2008; 14(5):844-52. PMC: 2327352. DOI: 10.1261/rna.939908. View

13.

Martinez-Beamonte R, Navarro M, Larraga A, Strunk M, Barranquero C, Acin S . Selection of reference genes for gene expression studies in rats. J Biotechnol. 2011; 151(4):325-34. DOI: 10.1016/j.jbiotec.2010.12.017. View

14.

Liang Y, Ridzon D, Wong L, Chen C . Characterization of microRNA expression profiles in normal human tissues. BMC Genomics. 2007; 8:166. PMC: 1904203. DOI: 10.1186/1471-2164-8-166. View

15.

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

16.

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

17.

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

18.

Ponton F, Chapuis M, Pernice M, Sword G, Simpson S . Evaluation of potential reference genes for reverse transcription-qPCR studies of physiological responses in Drosophila melanogaster. J Insect Physiol. 2011; 57(6):840-50. DOI: 10.1016/j.jinsphys.2011.03.014. View

19.

Derveaux S, Vandesompele J, Hellemans J . How to do successful gene expression analysis using real-time PCR. Methods. 2009; 50(4):227-30. DOI: 10.1016/j.ymeth.2009.11.001. View

20.

Bonefeld B, Elfving B, Wegener G . Reference genes for normalization: a study of rat brain tissue. Synapse. 2008; 62(4):302-9. DOI: 10.1002/syn.20496. View