Selection of stable reference genes in heat stressed peripheral blood mononuclear cells of tropically adapted Indian cattle and buffaloes

Kishore, Amit; Sodhi, Monika; Khate, Keviletsu; Kapila, Neha; Kumari, Parvesh; Mukesh, Manishi

doi:10.1016/j.mcp.2013.02.003

Cited by 30 publications

(22 citation statements)

References 26 publications

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“…In recent past, our groups has conducted number of similar Reference genes for qPCR studies across tissues of riverine buffaloes Reference genes for qPCR studies across tissues of riverine buffaloes studies to identify appropriate reference gene panels for application in transcriptional studies in Indian native cattle and riverine buffaloes. In one such study, β2M, RPS9 and RPS15a genes were identified as best RGs for heat stressed mononuclear cells of Indian cattle and buffaloes [19]; β2M, RPS23, RPL4 and EEF1A1 as most reliable RGs in heat stressed mammary explants and mammary epithelial cells of buffaloes [6,7]; RPL4, EEF1A1, ACTB and GAPDH genes were found to be most stable genes in milk derived mammary epithelial cells in Sahiwal cows during different lactation stages [30]. Similarly identification of stable reference genes for transcriptional studies in bulls distinctive in meat quality [5] and in buffalo muscle tissue [31] were also reported.…”

Section: Discussionmentioning

confidence: 99%

“…The RNA samples were stored at -80˚C till further use. First strand cDNA was synthesized using 100ng RNA as described in our previous studies [7,19] using the program: 25˚C for 5 min, 50˚C for 60 min, and 70˚C for 15 min. cDNA was then diluted 1:4 (v:v) with DNase/RNase free water.…”

Section: Total Rna Extraction and Cdna Synthesismentioning

confidence: 99%

“…Therefore, number of studies have successfully been conducted to identify least regulated and stably expressed RGs [9][10][11] across tissues of various species such as human, pig, sheep, bovines etc [12][13][14][15][16][17][18]. To our knowledge, not much information is available on set of suitable reference genes that can be used in lactation or physiological studies in riverine buffaloes (Bubalus bubalis) although few studies have been performed in specific cell types/tissues [19][20][21]. Considering the fact that choice of suitable reference/RGs genes is crucial for accurate expression profiling of target genes, this study presents the information on panel of appropriate RGs across 12 buffalo tissues.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2018

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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.

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Section: Discussionmentioning

confidence: 99%

Section: Total Rna Extraction and Cdna Synthesismentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

et al. 2018

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“…Kishore et al (2013) evaluated the expression stability of 11 commonly reported ICGs in heat stressed PBMCs of cattle and buffaloes based on stability measures. The analysis identified Beta-2 microglobulin (B2M) and ribosomal proteins (RPS9 and RPS15) as most stably expressed genes and were thus used for normalization of expression data of major HSP genes.…”

Section: Normalization and Data Analysismentioning

confidence: 99%

Peripheral blood mononuclear cells: a potential cellular system to understand differential heat shock response across native cattle (Bos indicus), exotic cattle (Bos taurus), and riverine buffaloes (Bubalus bubalis) of India

Kishore

Sodhi

Kumari

et al. 2014

Cell Stress and Chaperones

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Circulating leukocytes can be used as an effective model to understand the heat stress response of different cattle types and buffaloes. This investigation aimed to determine the temporal profile of HSPs (HSP40, HSP60, HSP70, and HSP90) expression in circulating peripheral blood mononuclear cells (PBMCs) of Murrah buffaloes, Holstein-Friesian (HF), and Sahiwal cows in response to sublethal heat shock at 42°C. The viability data indicated HF PBMCs to be the most affected to the heat shock, whereas Sahiwal PBMCs were least affected, indicating its better survivability during the heat stress condition. The qRT-PCR expression data showed significant increase in mRNA expression of the analyzed HSPs genes after heat stimuli to the PBMCs under in vitro condition. In each case, the HSPs were most upregulated at 2 h after the heat stress. Among the HSPs, HSP70 was relatively more expressed followed by HSP60 indicating the action of molecular chaperones to stabilize the native conformation of proteins. However, PBMCs from different cattle types and buffaloes showed difference in the extent of transcriptional response. The level of expression of HSPs throughout the time period of heat stress was highest in buffaloes, followed by HF and Sahiwal cows. The higher abundance of HSP70 mRNA at each time point after heat stress showed prolonged effect of heat stress in HF PBMCs. The data presented here provided initial evidence of transcriptional differences in PBMCs of different cattle types and buffaloes and warrant further research.

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“…In the present study, the SG and RB breeds appeared to have more genetic variants at HSP 90 than WF and AM breeds showing a greater polymorphisms in the former's than the latter. The detected genetic variants of HSP 90 gene could be interrogated as veritable genetic resource for improvement programme of thermo-tolerance, adaptability and survivability advantage to cope with wide range of thermal stress and environmental variations especially in the hot humid tropics (Schwerin et al, 2002a;Kishore et al, 2013) as well as disease tolerance and drug resistance of animals under thermal stress (Zhang et al, 2002b;Aufricht, 2005;Singh et al, 2006).…”

Section: Discussionmentioning

confidence: 99%

Evaluation of Polymorphisms at Heat Shock Protein 90 Gene by High Resolution Melting Assays for Potential Heat Tolerance among Nigerian Zebu Cattle Breeds

Onasanya¹,

Msalya

Thiruvenkadan

et al. 2020

American Journal of Animal and Veterinary Sciences

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Heat Shock Protein (HSP) 90 gene is a member of HSPs subfamily that act as molecular chaperons whenever animals come under thermal stress. The genes fulfill essential roles of providing cellular protection, immune response, protein synthesis, protein folding and unfolding, protection from cellular stress, inhibitory apoptosis and adaptation. This study was designed to analyze polymorphisms of HSP 90 and to evaluate their influence on heat tolerance among selected Nigerian zebu. The polymorphisms were also used to determine genetic relationship among the animals. About 450 bp of bovine HSP 90 including part of coding region in exon 3 was sequenced in 90 DNA samples representing four Nigerian zebu namely White Fulani (WF), Sokoto Gudali (SG), Red Bororo (RB) and Ambala (AM). Sequencing was done using an automated ABI-DNA Sequencer. Editing was accomplished using chromatogram analyses on SeqMan Ngen Tool. Rooted phylogenetic tree was constructed using MEGA 5.2 software. In total, 11 genetic variants were determined. Five of these (PRP, RED, ORG, LMN and YLO) were major variants detected in over 70% of the samples. Six (6) were classified as minor variants detected in two breeds or less and in 29.1% of the samples. The GRN and NBL were only detected in RB and SG breeds respectively. We found a shared homology and common ancestral lineage among the breeds. Furthermore, the genetic structure of Nigerian zebu has a common clade architecture to those of goats, sheep, yak, buffalo, camel, horse and other taurines. The gene is conserved among wide range of animals and as such it can serve as one of bio-markers for selection and breeding programmes for thermotolerance in wide range of livestock animals under thermal stress. The variant groups could be further interrogated for possible specific effects on thermotolerance performance of zebu in hot tropical environments.

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Selection of stable reference genes in heat stressed peripheral blood mononuclear cells of tropically adapted Indian cattle and buffaloes

Cited by 30 publications

References 26 publications

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

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

Peripheral blood mononuclear cells: a potential cellular system to understand differential heat shock response across native cattle (Bos indicus), exotic cattle (Bos taurus), and riverine buffaloes (Bubalus bubalis) of India

Evaluation of Polymorphisms at Heat Shock Protein 90 Gene by High Resolution Melting Assays for Potential Heat Tolerance among Nigerian Zebu Cattle Breeds

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