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. 2013 Jan 28:2013:735053.
doi: 10.5402/2013/735053. eCollection 2013.

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

Neha Kapila  1 , Amit Kishore  2 , Monika Sodhi  2 , Ankita Sharma  2 , Pawan Kumar  3 , A K Mohanty  4 , Tanushri Jerath  2 , M Mukesh  2
Affiliations

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

Neha Kapila et al. ISRN Biotechnol. .

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.

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Figures

Figure 1
Figure 1
Overall expression pattern of 16 genes evaluated in heat-stressed MECs. The data is represented as qPCR cycle threshold (Ct) values of each gene in the box and whisker diagram. The median is shown as a dashed line across the box. The boxes represent median and 1st and 3rd quartiles ranges, while whiskers indicate maximum and minimum values.
Figure 2
Figure 2
Average expression stability measures (M value) for reference genes.
Figure 3
Figure 3
Determination of optimal number of reference genes for normalization by calculation of pairwise variation (V) of normalization factor ratios for different number of genes.
Figure 4
Figure 4
Bar plot showing gene variability in 16 candidate reference genes by NormFinder.
See this image and copyright information in PMC

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