Selection of suitable reference genes for normalization of quantitative RT-PCR (RT-qPCR) expression data across twelve tissues of riverine buffaloes (Bubalus bubalis)

Abstract

Selection of reference genes has become an integral step in any real time quantitative PCR (RT-qPCR) based expression studies. The importance of this study stems from the fact that riverine buffaloes are major dairy species of Indian sub-continent and the information generated here will be of great interest to the investigators engaged in functional genomic studies of this important livestock species. In this study, an effort was made to evaluate a panel of 10 candidate reference genes (glyceraldehyde 3-phosphate dehydrogenase (GAPDH), beta- actin (ACTB), ubiquitously expressed transcript (UXT), ribosomal protein S15 (RPS15), ribosomal protein L-4 (RPL4), ribosomal protein S9 (RPS9), ribosomal protein S23 (RPS23), hydroxymethylbilane synthase (HMBS), β2 Microglobulin (β2M) and eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) across 12 tissues (mammary gland, kidney, spleen, liver, heart, intestine, ovary, lung, muscle, brain, subcutaneous fat and testis) of riverine buffaloes. In addition to overall analysis, tissue wise evaluation of expression stability of individual RG was also performed. Three different algorithms provided in geNorm, NormFinder and BestKeeper softwares were used to evaluate the stability of 10 potential reference genes from different functional classes. The M-value given by geNorm ranged from 0.9797 (RPS9 and UXT) to 1.7362 (RPS15). From the most stable to the least stable, genes were ranked as: UXT/RPS9> RPL4> RPS23> EEF1A1> ACTB> HMBS> GAPDH> B2M> RPS15. While NormFinder analysis ranked the genes as: UXT> RPS23> RPL4> RPS9> EEF1A1> HMBS> ACTB> β2M> GAPDH> RPS15. Based on the crossing point SD value and range of fold change expression, BestKeeper analysis ranked the genes as: RPS9> RPS23/UXT> RPL4> GAPDH> EEF1A1> ACTB> HMBS> β2M> RPS15. Overall the study has identified RPS23, RPS9, RPL4 and UXT genes to be the most stable and appropriate RGs that could be utilized for normalization of transcriptional data in various tissues of buffaloes. This manuscript thus provide useful information on panel of reference genes that could be helpful for researchers conducting functional genomic studies in riverine buffaloes.

Fig 1. Expression levels of individual candidate RGs.

The data is presented as quantification cycle (Cq) values of each gene in the box-whisker diagram. The median is shown as a line across the box while whiskers indicate maximum and minimum values.

Fig 2. Ranking of RGs based on average expression stability values (M values).

Fig 3. The pairwise variation (V) analysis of candidate RGs for determining the optimal number of genes for normalization across 12 buffalo tissues.

Fig 4. Tissue wise expression stability value (M value) of candidate RGs by geNorm analysis.

Fig 5. Pairwise variation (V) analysis of 10 RGs to determine optimal number of reference genes for normalization of qPCR data in individual buffalo tissue.

Fig 6. Bar plot showing gene variability across 10 RGs by NormFinder.

Fig 7. Inter-group variation analysis of RGs across different tissues.

Fig 8. Intra-group variation analysis of candidate RGs in each tissue.

Fig 9. Expression stability of 4 selected RGs (RPL4, RPS23, RPS9, UXT) across buffalo tissues.

MG-Mammary Gland, KID-Kidney, SPL-Spleen, LIV-Liver, HRT-Heart, INT-Intestine, OVA-Ovary, LUNG-Lung, MUS-Muscle, BRN-Brain, S. FAT-Subcutaneous fat, TES-Testis.