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Development and validation of most efficient RNA isolation method from buffalo bull spermatozoa

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Abstract

Background

Being highly fragmented and low in concentration, isolation of good quality RNA from sperm cells is a big challenge. Attempts have been made to evaluate various sperm RNA isolation methods from purified buffalo bull sperm cells.

Methods

Both, non-membrane and membrane-based methods have been evaluated for isolating RNA from Murrah buffalo sperms and compared for their respective efficacies. The traditional TRIzol, TRIzol-heat lysed (H-TRIzol) and cocktail of TCEP-RLT lysis buffer (Qiagen RNeasy mini kit)-TRIzol (C-TRIzol) based isopropanol isolation methods have been evaluated.

Results

H-TRIzol yielded best results among conventional methods. The combined T-RLT RNA isolation protocol yielded best quality and quantity compared to other membrane-based methods, due to high lytic property of cocktail of lysis reagents, necessary for complete breakdown of sperm membrane and RNA binding membrane for RNA isolation. Combined lysis performed by treatment with RLT-T and T-RLT differing in order of reagents used were also evaluated. T-RLT combination giving better results compared to RLT-T due to high gDNA contamination and membrane clogging in later protocol steps.

Conclusion

Overall, in terms of total RNA quantity and quality per million spermatozoa, the heat-lysed TRIzol method (H-TRIzol) performs best among RNA separation techniques employed and is also quite easy to perform. This comparative evaluation of sperm RNA isolation protocols can be useful in deciding the best protocol for isolation of good quality and high concentration sperm RNA from buffalo semen, for transcriptome and other downstream studies.

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Data Availability

The datasets generated during and/or analysed during the current study are not publicly available but are available from the corresponding author on reasonable request.

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Acknowledgements

Receiving of Senior Research Fellowship of DBT by first author RV (DBT/2018/NDRI/996) is also acknowledged.

Funding

This work was supported by the Department of Biotechnology, Govt. of India, (Grant number BT/PR30439/AAQ/1/743/ 2019).

Author information

Authors and Affiliations

  1. Animal Biotechnology Division, ICAR-National Bureau of Animal Genetic Resources, Karnal, Haryana, 132001, India

    Rashi Vasisth, Ankita Gurao, Namita Kumari, Gautam Kumar, Anurag Kumar, Mahesh Shivanand Dige, Manishi Mukesh, Rajeev Anand Kumar Aggarwal & Ranjit Singh Kataria

  2. ICAR-National Dairy Research Institute, Karnal, Haryana, 132001, India

    Rashi Vasisth, Karpenahalli Ranganatha Sriranga & Pawan Singh

Authors
  1. Rashi Vasisth
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  2. Ankita Gurao
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  11. Ranjit Singh Kataria
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Contributions

Rashi Vasisth, Ankita Gurao, Namita Kumari: investigation, methodology. Gautam Kumar, Anurag Kumar, Karpenahalli Ranganatha Sriranga: samples and animal data collection. Mahesh Shivanand Dige, Rajeev Anand Kumar Aggarwal: sample processing. Manishi Mukesh, Pawan Singh and Ranjit Singh Kataria: conceptualization of experiments, data analysis, review and editing.

Corresponding author

Correspondence to Ranjit Singh Kataria.

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Vasisth, R., Gurao, A., Kumari, N. et al. Development and validation of most efficient RNA isolation method from buffalo bull spermatozoa. Mol Biol Rep 50, 6717–6727 (2023). https://doi.org/10.1007/s11033-023-08593-0

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