Selection and Validation of Suitable Reference Genes for QPCR Gene Expression Analysis in Goats and Sheep Under Peste Des Petits Ruminants Virus (PPRV), Lineage IV Infection

Overview

Journal Sci Rep

Specialty Science

Date 2018 Oct 31

PMID 30374051

Citations 10

Authors

Amit Ranjan Sahu

Sajad Ahmad Wani

Shikha Saxena

Kaushal Kishor Rajak

Dheeraj Chaudhary

Aditya Prasad Sahoo

Alok Khanduri

Aruna Pandey

Piyali Mondal

Waseem Akram Malla

Raja Ishaq Nabi Khan

Ashok Kumar Tiwari

Bina Mishra

D Muthuchelvan

Bishnu Prasad Mishra

Raj Kumar Singh

Ravi Kumar Gandham

Affiliations

Soon will be listed here.

Abstract

Identification of suitable candidate reference genes is an important prerequisite for validating the gene expression data obtained from downstream analysis of RNA sequencing using quantitative real time PCR (qRT-PCR). Though existence of a universal reference gene is myth, commonly used reference genes can be assessed for expression stability to confer their suitability to be used as candidate reference genes in gene expression studies. In this study, we evaluated the expression stability of ten most commonly used reference genes (GAPDH, ACTB, HSP90, HMBS, 18S rRNA, B2M, POLR2A, HPRT1, ACAC, YWHAZ) in fourteen different Peste des petits ruminants virus (PPRV) infected tissues of goats and sheep. RefFinder and RankAggreg software were used to deduce comprehensive ranking of reference genes. Our results suggested HMBS and B2M in goats and HMBS and HPRT1 in sheep can be used as suitable endogenous controls in gene expression studies of PPRV infection irrespective of tissues and condition as a whole, thus eliminating the use of tissue specific/ condition specific endogenous controls. We report for the first time suitable reference genes for gene expression studies in PPRV infected tissues. The reference genes determined here can be useful for future studies on gene expression in sheep and goat infected with PPRV, thus saving extra efforts and time of repeating the reference gene determination and validation.

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References

Bonnet M, Bernard L, Bes S, Leroux C . Selection of reference genes for quantitative real-time PCR normalisation in adipose tissue, muscle, liver and mammary gland from ruminants. Animal. 2013; 7(8):1344-53. DOI: 10.1017/S1751731113000475. View

Wang Z, Gerstein M, Snyder M . RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet. 2008; 10(1):57-63. PMC: 2949280. DOI: 10.1038/nrg2484. View

Hellemans J, Vandesompele J . Selection of reliable reference genes for RT-qPCR analysis. Methods Mol Biol. 2014; 1160:19-26. DOI: 10.1007/978-1-4939-0733-5_3. View

Banyard A, Parida S, Batten C, Oura C, Kwiatek O, Libeau G . Global distribution of peste des petits ruminants virus and prospects for improved diagnosis and control. J Gen Virol. 2010; 91(Pt 12):2885-97. DOI: 10.1099/vir.0.025841-0. View

McNeill R, Miller N, Kerin M . Evaluation and validation of candidate endogenous control genes for real-time quantitative PCR studies of breast cancer. BMC Mol Biol. 2007; 8:107. PMC: 2211316. DOI: 10.1186/1471-2199-8-107. View

Taylor S, Wakem M, Dijkman G, Alsarraj M, Nguyen M . A practical approach to RT-qPCR-Publishing data that conform to the MIQE guidelines. Methods. 2010; 50(4):S1-5. DOI: 10.1016/j.ymeth.2010.01.005. View

Baron J, Bin-Tarif A, Herbert R, Frost L, Taylor G, Baron M . Early changes in cytokine expression in peste des petits ruminants disease. Vet Res. 2014; 45:22. PMC: 3941941. DOI: 10.1186/1297-9716-45-22. View

Harty R, Pitha P, Okumura A . Antiviral activity of innate immune protein ISG15. J Innate Immun. 2009; 1(5):397-404. PMC: 2725329. DOI: 10.1159/000226245. View

Rajkumar A, Qvist P, Lazarus R, Lescai F, Ju J, Nyegaard M . Experimental validation of methods for differential gene expression analysis and sample pooling in RNA-seq. BMC Genomics. 2015; 16:548. PMC: 4515013. DOI: 10.1186/s12864-015-1767-y. View

10.

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

11.

Ye J, Coulouris G, Zaretskaya I, Cutcutache I, Rozen S, Madden T . Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics. 2012; 13:134. PMC: 3412702. DOI: 10.1186/1471-2105-13-134. View

12.

Marioni J, Mason C, Mane S, Stephens M, Gilad Y . RNA-seq: an assessment of technical reproducibility and comparison with gene expression arrays. Genome Res. 2008; 18(9):1509-17. PMC: 2527709. DOI: 10.1101/gr.079558.108. View

13.

Radonic A, Thulke S, Mackay I, Landt O, Siegert W, Nitsche A . Guideline to reference gene selection for quantitative real-time PCR. Biochem Biophys Res Commun. 2004; 313(4):856-62. DOI: 10.1016/j.bbrc.2003.11.177. View

14.

Serrano M, Moreno-Sanchez N, Gonzalez C, Marcos-Carcavilla A, Van Poucke M, Calvo J . Use of Maximum Likelihood-Mixed Models to select stable reference genes: a case of heat stress response in sheep. BMC Mol Biol. 2011; 12:36. PMC: 3175163. DOI: 10.1186/1471-2199-12-36. View

15.

Mortazavi A, Williams B, McCue K, Schaeffer L, Wold B . Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods. 2008; 5(7):621-8. DOI: 10.1038/nmeth.1226. View

16.

Han X, Lu M, Chen Y, Zhan Z, Cui Q, Wang Y . Selection of reliable reference genes for gene expression studies using real-time PCR in tung tree during seed development. PLoS One. 2012; 7(8):e43084. PMC: 3422230. DOI: 10.1371/journal.pone.0043084. View

17.

Pihur V, Datta S, Datta S . RankAggreg, an R package for weighted rank aggregation. BMC Bioinformatics. 2009; 10:62. PMC: 2669484. DOI: 10.1186/1471-2105-10-62. View

18.

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

19.

Najafpanah M, Sadeghi M, Bakhtiarizadeh M . Reference genes selection for quantitative real-time PCR using RankAggreg method in different tissues of Capra hircus. PLoS One. 2013; 8(12):e83041. PMC: 3865150. DOI: 10.1371/journal.pone.0083041. View

20.

Patel A, Rajak K, Balamurugan V, Sen A, Sudhakar S, Bhanuprakash V . Cytokines expression profile and kinetics of Peste des petits ruminants virus antigen and antibody in infected and vaccinated goats. Virol Sin. 2012; 27(4):265-71. PMC: 8218146. DOI: 10.1007/s12250-012-3240-2. View