Dataset Information

Identification of Appropriate Reference Genes for qRT-PCR Analysis of Heat-Stressed Mammary Epithelial Cells in Riverine Buffaloes (Bubalus bubalis).

ABSTRACT: Gene expression studies require appropriate normalization methods for proper evaluation of reference genes. To date, not many studies have been reported on the identification of suitable reference genes in buffaloes. The present study was undertaken to determine the panel of suitable reference genes in heat-stressed buffalo mammary epithelial cells (MECs). Briefly, MEC culture from buffalo mammary gland was exposed to 42?°C for one hour and subsequently allowed to recover at 37?°C for different time intervals (from 30?m to 48?h). Three different algorithms, geNorm, NormFinder, and BestKeeper softwares, were used to evaluate the stability of 16 potential reference genes from different functional classes. Our data identified RPL4, EEF1A1, and RPS23 genes to be the most appropriate reference genes that could be utilized for normalization of qPCR data in heat-stressed buffalo MECs.

SUBMITTER: Kapila N

PROVIDER: S-EPMC4393032 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Identification of Appropriate Reference Genes for qRT-PCR Analysis of Heat-Stressed Mammary Epithelial Cells in Riverine Buffaloes (Bubalus bubalis).

Kapila Neha N Kishore Amit A Sodhi Monika M Sharma Ankita A Kumar Pawan P Mohanty A K AK Jerath Tanushri T Mukesh M M

ISRN biotechnology 20130128

Gene expression studies require appropriate normalization methods for proper evaluation of reference genes. To date, not many studies have been reported on the identification of suitable reference genes in buffaloes. The present study was undertaken to determine the panel of suitable reference genes in heat-stressed buffalo mammary epithelial cells (MECs). Briefly, MEC culture from buffalo mammary gland was exposed to 42 °C for one hour and subsequently allowed to recover at 37 °C for different ...[more]

PMID: 25937980

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Project description:The present study aims to identify the heat responsive genes and biological pathways in heat stressed buffalo mammary epithelial cells (MECs). The primary mammary epithelial cells of riverine buffalo were exposed to thermal stress at 42°C for one hour. The cells were subsequently allowed to recover at 37°C and harvested at different time intervals (30 min to 48 h) along with control samples (un-stressed). In order to assess the impact of heat stress in buffalo MECs, several in-vitro cellular parameters (lactate dehydrogenase activity, cell proliferation assay, cellular viability, cell death and apoptosis) and transcriptional studies were conducted. The heat stress resulted in overall decrease in cell viability and cell proliferation of MECs while induction of cellular apoptosis and necrosis. The transcriptomic profile of heat stressed MECs was generated using Agilent 44 K bovine oligonucleotide array and at cutoff criteria of ≥3-or ≤3 fold change, a total of 153 genes were observed to be upregulated while 8 genes were down regulated across all time points post heat stress. The genes that were specifically up-regulated or down-regulated were identified as heat responsive genes. The upregulated genes in heat stressed MECs belonged to heat shock family viz., HSPA6, HSPB8, DNAJB2, HSPA1A. Along with HSPs, genes like BOLA, MRPL55, PFKFB3, PSMC2, ENDODD1, ARID5A, and SENP3 were also upregulated. Microarray data revealed that the heat responsive genes belonged to different functional classes viz., chaperons; immune responsive; cell proliferation and metabolism related. Gene ontology analysis revealed enrichment of several biological processes like; cellular process, metabolic process, response to stimulus, biological regulation, immune system processes and signaling. The transcriptome analysis data was further validated by RT-qPCR studies. Several HSP (HSP40, HSP60, HSP70, HSP90, and HSPB1), apoptotic (Bax and Bcl2), immune (IL6, TNFα and NF-kβ) and oxidative stress (GPX1 and DUSP1) related genes showed differential expression profile at different time points post heat stress. The transcriptional data strongly indicated the induction of survival/apoptotic mechanism in heat stressed buffalo MECs. The overrepresented pathways across all time points were; electron transport chain, cytochrome P450, apoptosis, MAPK, FAS and stress induction of HSP regulation, delta Notch signaling, apoptosis modulation by HSP70, EGFR1 signaling, cytokines and inflammatory response, oxidative stress, TNF-alpha and NF- kB signaling pathway. The study thus identified several genes from different functional classes and biological pathways that could be termed as heat responsive in buffalo MEC. The responsiveness of buffalo MECs to heat stress in the present study clearly suggested its suitability as a model to understand the modulation of buffalo mammary gland expression signature in response to environmental heat load.

Project description: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), ?2 Microglobulin (?2M) 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> ?2M> 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> ?2M> 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.

Project description:Traumatic pericarditis (TP) remains a serious problem facing bovine producers; particularly in the developing countries; causing severe economic losses. This study was carried out on 47 buffaloes including; control buffaloes (n = 20) and buffaloes with TP (n = 27) at Assiut governorate, Egypt. All animals were subjected to thorough clinical examination, whole blood profiling, blood serum biochemical assays, and radiographic and ultrasonographic examinations. The recorded ultrasonographic findings included; the contractility, contour and shape of the reticulum, shape and size of the heart and spleen involvement. All data were statistically analysed. Buffaloes with TP showed lymphocytic leucocytosis, increase in serum activities of AST, hyperproteinaemia, hypoalbuminaemia and hyperglobulinaemia. Radio-opaque metal foreign bodies, cardiomegaly and loss of the normal cardiac shape and contour were the most common radiographic findings in the diseased buffaloes. Ultrasonographically, the diseased buffaloes showed either acute pericarditis (n = 10) or chronic suppurative pericarditis (n = 17). Complete cessation of reticular contractions (0/3 min) and displacement of the reticulum from the diaphragm by a distance about 2.6-4.5 cm were the most common ultrasonographic findings of both types of pericarditis. In addition, the reticulum had uneven contour and the heart showed characteristic changes in its size, shape and contractility that was either accelerated or reduced. Reticular abscesses and peritoneal effusions were also imaged in all diseased buffaloes. In acute TP; the affected heart was enlarged with strong and clear cardiac contractions. Accumulation of hypoechoic fluids interspersed with echogenic deposits of fibrin within heart tissue and pericardium was observed. In chronic suppurative pericarditis, cardiomegaly, thickening of the cardiac wall and loss of the characteristic recognisable heart shape were the common ultrasonographic findings. Accumulation of hypoechoic fluids (usually pus) interspersing with echogenic deposits of fibrin within the pericardium, ventricles, atria and valves was also observed. In conclusion, ultrasonography with the aids of other diagnostic tools such as clinical findings, laboratory analysis and radiography has a high efficacy in evaluation of diseased buffaloes with TP.

Dataset Information

Identification of Appropriate Reference Genes for qRT-PCR Analysis of Heat-Stressed Mammary Epithelial Cells in Riverine Buffaloes (Bubalus bubalis).

Publications

Identification of Appropriate Reference Genes for qRT-PCR Analysis of Heat-Stressed Mammary Epithelial Cells in Riverine Buffaloes (Bubalus bubalis).

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