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RT-LAMP assay: an alternative approach for profiling of bovine heat shock protein 70 gene in PBMC cultured model

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Abstract

The purpose of this study is to develop a novel Reverse Transcriptase Loop-mediated isothermal amplification (RT-LAMP) based assay for in vitro profiling of heat shock protein 70 (Hsp70) in bovine peripheral blood mononuclear cell (PBMC) culture model utilizing the absorbance level of magnesium pyrophosphate—a by-product of LAMP reaction. A set of bovine Hsp70 specific RT-LAMP primers were designed to detect the differential absorbance level of magnesium pyrophosphate by-product which signifies the degree of Hsp70 amplification from cDNA of thermally induced cultured cells at different recovery periods. The study revealed significant (P < 0.05) correlation between absorbance level and the fold change of Hsp70 transcripts at different kinetic intervals of heat stress recovery in bovine PBMC cell culture models. RT-LAMP based absorbance assay can be used as an indicator to measure the degree of bovine Hsp70 transcripts produced during thermal stress and can be used as an alternative to the traditional Real time PCR assay. Developed RT-LAMP assay can be used as a cost-effective method for profiling of bovine HSP70 gene.

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Acknowledgements

The authors are thankful to the Director, ICAR-CIRC Meerut for providing necessary facilities to carry out the present research. Authors also acknowledge the Science and Engineering Research Board, Government of India for providing financial support under the project YSS/2014/000279 to RD.

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Authors and Affiliations

  1. Molecular Genetics Laboratory, ICAR-Central Institute for Research on Cattle, Meerut, 250001, Uttar Pradesh, India

    Gyanendra Singh Sengar, Rajib Deb, T. V. Raja, Umesh Singh, V. Bhanuprakash, R. R. Alyethodi, Sushil Kumar, Rani Alex & Rani Singh

  2. Department of Cellular & Molecular Engineering, Sam Higginbottom University of Agriculture, Technology and Sciences, Allahabad, 211007, Uttar Pradesh, India

    Gyanendra Singh Sengar, Rajiv Kant & Preetam Verma

  3. ICAR-Indian Veterinary Research Institute, Izatnagar, 243122, Uttar Pradesh, India

    Soumendu Chakraborty

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  1. Gyanendra Singh Sengar
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  2. Rajib Deb
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  3. T. V. Raja
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  4. Umesh Singh
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  5. Rajiv Kant
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  6. V. Bhanuprakash
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  7. R. R. Alyethodi
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  8. Sushil Kumar
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  9. Preetam Verma
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  10. Soumendu Chakraborty
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  11. Rani Alex
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  12. Rani Singh
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Corresponding author

Correspondence to Rajib Deb.

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The authors declare that they have no conflict of interest.

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Institutional animal ethical permission has been taken for collection of blood samples.

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Sengar, G.S., Deb, R., Raja, T.V. et al. RT-LAMP assay: an alternative approach for profiling of bovine heat shock protein 70 gene in PBMC cultured model. Mol Biol Rep 44, 281–287 (2017). https://doi.org/10.1007/s11033-017-4108-1

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  • DOI: https://doi.org/10.1007/s11033-017-4108-1

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