Computational protocol: Transcriptome Analysis of Circulating PBMCs to Understand Mechanism of High Altitude Adaptation in Native Cattle of Ladakh Region

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Protocol publication

[…] The slides were scanned immediately after washing on Agilent DNA Microarray Scanner (G2505B) using one colour scan setting. The signal intensities were checked from the digit images using Agilent Feature Extractor Software Version 9.5 (Agilent Technologies). The microarray raw data files in.txt format were imported to GeneSpring GX 13.1 (Agilent Technologies) and subjected for quantile normalization which is highly effective in reducing variation between arrays. Normalized data was analysed for statistically significant gene expression differences between Ladakhi cows and Sahiwal cows to the fold change statistic that uses overall gene expression variation to calculate a gene-specific variance. To keep the number of false positives to alpha = 0.05, a false discovery rate adjustment was used. Genes that were statistically significant in expression between Ladakhi cows and Sahiwal cows were grouped according to fold expression (up- vs. down-regulation). The data was further subjected to hierarchical clustering and k-means clustering based on entities (genes), gene ontology enrichment analysis and pathway analysis. [...] The microarray data was further validated by comparing the expression of some of the up-regulated hypoxia related genes such as HIF-1α, EPAS-1, VEGF-A, ECE-1, NOS2, GLUT-1, HK2 and heat shock proteins (HSP27, HSP70 & HSP90) in cattle types from high altitude region and tropical region. For this a total of 30 PBMC samples were selected; 5 each of Ladakhi cows (LAC), Holstein Frisian crosses (HFX), Jersey cows (JYC) from high altitude region; and Sahiwal cows (SAC), Karan Fries cows (KFC), Holstein Friesian cows (HFC) from tropical region using qPCR. For optimal gene expression analysis, qPCR data of target genes was normalized using a total of 10 previously known candidate reference genes from different functional categories viz., GAPDH, RPL4, EEF1A1, RPS9, HPRT, UXT, HMBS, B2M, RPS15 and ACTB. The primers for each of the ICGs and target genes were either selected from literature or designed using Primer express 3.0 software (Applied Biosystem). Primer details for all the ICGs and target genes are provided as supplementary information (Tables  and ). The accuracy of primer pairs was also ensured by the presence of a unique peak during the dissociation step at the end of qPCR cycle. The qPCR was performed using Light Cycler 480 instrument (Roche, Germany) as described in our previous reports. The data was acquired using the ‘second derivative maximum’ method as computed by the Light Cycler Software 3.5 (Roche Diagnostics) and subjected for subsequent analysis. [...] qPCR data of target genes in high altitude and tropically adapted cattle was normalized by selecting best suited ICGs utilizing geNorm, NormFinder and Best keeper softwares–.The mean value was calculated using relative quantification 2−ΔΔCT method. The differences between groups were tested by Tukey’s Multiple Comparison Test. P values less than 0.05 and 0.01 were considered significant. Statistical test was performed using GraphPad PRISM version 5.0 (La Jolla, CA, USA). […]

Pipeline specifications

Software tools GeneSpring GX, Primer Express, NormFinder
Applications Gene expression microarray analysis, qPCR
Organisms Bos taurus