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

Amit Ranjan Sahu,Alok Khanduri,Dheeraj Chaudhary,Piyali Mondal,Kaushal Kishor Rajak,Raj Kumar Singh,Shikha Saxena,Ashok Kumar Tiwari,D Muthuchelvan,Sajad Ahmad Wani,Ravi Kumar Gandham,Aditya Prasad Sahoo,Waseem Akram Malla,Raja Ishaq Nabi Khan,Aruna Pandey,Bina Mishra,Bishnu Prasad Mishra

doi:10.1038/s41598-018-34236-7

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.

Highlights

In the era of high throughput sequencing, RNA–Sequencing (RNA-Seq) has been widely applied to evaluate global gene expression levels and composition[1,2,3]
The tissue samples were found positive for Peste des petits ruminants virus (PPRV) by sandwich ELISA, N gene based RT-PCR and quantitative RT-PCR (qRT-PCR) (Supplementary Fig. S5) and histopathology and immunohistochemistry
Expression of PPRV - N gene was detected in all the infected tissues of goats and sheep by qRT-PCR (Supplementary Figs S5A and S5B)

Summary

Introduction

In the era of high throughput sequencing, RNA–Sequencing (RNA-Seq) has been widely applied to evaluate global gene expression levels and composition[1,2,3]. The use of an invalidated reference gene in normalization leads to unreliable conclusions especially when used with tissue samples[15,18,23] This warrants for a need to identify suitable reference gene(s) for normalization for every gene expression experiment to do away with the hurdles in qRT-PCR24. In our study we used a panel of ten reference genes viz. GAPDH (Glyceraldehyde-3-phosphate dehydrogenase), 18S rRNA (18S ribosomal RNA), B2M (β 2 microglobulin), HSP 90 (heat shock protein 90), ACAC-alpha (Acetyl coenzyme carboxylase alpha), HMBS (Hydroxymethylbilane synthase), YWHAZ (Tyrosine 3-monooxygenase activation protein zeta polypeptide), POLR2A (Polymerase[32] II (DNA directed) polypeptide A), ACTB (beta actin) and HPRT1 (Hypoxanthin Phosphoribosyl transferase 1) in fourteen different tissues obtained from healthy and PPRV infected goats and sheep to identify the best possible reference gene(s) for qRT-PCR normalization. We recommend different sets of reference genes based on the experimental condition

Methods

Results

Conclusion