Evaluation of housekeeping genes as references for quantitative real-time PCR analysis of gene expression in the murrel Channa striatus under high-temperature stress
Quantitative real-time polymerase chain reaction is the most advanced method of quantifying gene expression studies; however, the significance of the obtained results strongly depends on the normalization of the data to compensate for differences between the samples. In the present study, expression analysis of six different constitutively expressed genes viz. 18S ribosomal RNA, glyceraldehyde-3-phosphate dehydrogenase (gapdh), beta actin (βactin), ribosomal binding protein L13, tubulin and TATA-box-binding protein (tbp) were carried out to test their efficacy as reference genes in three different tissues, namely liver, gill and muscle of murrel Channa striatus exposed to high temperature for variable time periods. The stability and suitability of the genes were determined by using bioinformatic tools: GeNorm, NormFinder and BestKeeper. Based on the results, tub/βactin could be used as the reference genes for liver and gill tissues and βactin/gapdh could be the reference genes for muscle tissues in Channa striatus under both short- and long-term thermal stress.
KeywordsReference genes Quantitative real-time PCR Normalization Channa striatus Thermal stress
- IPCC (2007) Fourth Assessment Report—Climate Change, Synthesis Report. Geneva, SwitzerlandGoogle Scholar
- Kapila N, Kishore A, Sodhi M, Sharma A, Kumar P, Mohanty AK, Jerath T, Mukesh M (2013) Identification of appropriate reference genes for qRT-PCR analysis of heat-stressed mammary epithelial cells in riverine buffaloes (Bubalus bubalis). ISRN Biotechnol 2013:735053PubMedCentralCrossRefPubMedGoogle Scholar
- Mohanty S, Mahanty A, Yadav RP, Purohit GK, Mohanty BN, Mohanty BP (2014) Atri hot spring—a natural ecosystem for global warming research. Int J Geol Earth Environ Sci 4:85–90Google Scholar
- Silva RLO, Silva MD, Neto JRCF, de Nardi CH, Moutinho S (2014) Validation of novel reference genes for reverse transcription quantitative real-time PCR in drought-stressed sugarcane. Sci World J 2014:357052Google Scholar
- Vandesompele J, Kubista M, Pfaffl MW (2009) Reference gene validation software for improved normalization. In: Logan J, Edwards K, Saunders N, Norfolk UK (eds) Real-time PCR: current technology and applications. Caister Academic Press, England, pp 47–64Google Scholar