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Evaluation of housekeeping genes as references for quantitative real-time PCR analysis of gene expression in the murrel Channa striatus under high-temperature stress

Abstract

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.

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Acknowledgments

This work was funded by the Indian Council of Agricultural Research under the National Fund for Basic, Strategic and Frontier Application Research in Agriculture (NFBSFARA) (currently renamed National Agricultural Science Fund, (NASF) (Project #AS: 2001) to BPM, SM. GKP and AM are Senior Research Fellows. The authors are thankful to the Director, KIIT School of Biotechnology, Bhubaneswar, and Director, ICAR-Central Inland Fisheries Research Institute, Barrackpore, for the facilities and encouragement.

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Correspondence to Bimal Prasanna Mohanty or Sasmita Mohanty.

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Electronic supplementary material

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10695_2015_123_MOESM1_ESM.pdf

Supplementary data 1 NCBI sequence information used for designing primers for amplification of different genes. Supplementary material 1 (PDF 24 kb)

10695_2015_123_MOESM2_ESM.pdf

Supplementary data 2 Standard curves showing (A) tbp, (B) βactin, (C) gapdh, (D) rbpl13, (E) tub, (F) 18SrRNA. Supplementary material 2 (PDF 293 kb)

10695_2015_123_MOESM3_ESM.pdf

Supplementary data 3 Descriptive statistics of the six candidate reference genes based on their cycle threshold (Ct) values as calculated by BestKeeper. Supplementary material 3 (PDF 35 kb)

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Purohit, G.K., Mahanty, A., Mohanty, B.P. et al. Evaluation of housekeeping genes as references for quantitative real-time PCR analysis of gene expression in the murrel Channa striatus under high-temperature stress. Fish Physiol Biochem 42, 125–135 (2016). https://doi.org/10.1007/s10695-015-0123-0

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  • DOI: https://doi.org/10.1007/s10695-015-0123-0

Keywords

  • Reference genes
  • Quantitative real-time PCR
  • Normalization
  • Channa striatus
  • Thermal stress