Present address: Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh, India.
Evaluating suitable internal control genes for transcriptional studies in heat-stressed mammary explants of buffaloes
Version of Record online: 29 MAY 2012
© 2012 Blackwell Verlag GmbH
Journal of Animal Breeding and Genetics
Volume 130, Issue 2, pages 106–117, April 2013
How to Cite
Sodhi, M., Kishore, A., Khate, K., Kapila, N., Mishra, B.P., Kataria, R.S., Mohanty, A.K., Varshney, N. and Mukesh, M. (2013), Evaluating suitable internal control genes for transcriptional studies in heat-stressed mammary explants of buffaloes. Journal of Animal Breeding and Genetics, 130: 106–117. doi: 10.1111/j.1439-0388.2012.01004.x
- Issue online: 17 MAR 2013
- Version of Record online: 29 MAY 2012
- Received: 7 September 2011; accepted: 26 March 2012
- Internal control genes;
- mammary gland;
- riverine buffalo
It is now a well-accepted notion that each new experimental design requires proper evaluation of internal control genes (ICGs) for accurate normalization of expression data. In riverine buffaloes, till date no appropriate ICG has been reported for studying transcriptional response under any of the physiological stressful condition. The objective here was to test 16 well-known reference genes from different functional categories that could serve as suitable ICG during heat stress studies in buffalo mammary tissue. Briefly, the mammary explants were exposed to 45°C for 1 h and subsequently allowed to recover at 37°C for different time points (2–24 h). Three software programs, geNorm, Normfinder and BestKeeper, were used to measure gene transcript stability. RPL22 was excluded because of weak amplification and unacceptable PCR efficiency. Except GAPDH, all other genes showed expression stability within the acceptable range (<1.5). RPL4, B2M, RPS23 and EEF1A1 genes were found to be most stably expressed while GAPDH and ACTB showed least stability. The BestKeeper analysis identified high correlation for RPL4 (r = 0.953) and EEF1A1 (r = 0.914) with BestKeeper index. Based on the present findings, it could be suggested that geometric average of RPL4, B2M, RPS23 and EEF1A1 would provide accurate normalization to transcriptional data of buffalo mammary explant in response to heat stress.