Modified TRIzol Method for extraction of Good quality RNA from Spikes of Black Pepper (Piper nigrum L.)

Author: Manjushri Dinkar Dongare, Swapna Alex, Soni K.B., Sindura K.P., Deepa S. Nair, Roy Stephen and Elizabeth Jose

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Abstract

Obtaining high quality RNA is a basic requirement for plant molecular biology, genetics, and physiological studies. High-quality RNA isolation from spikes of Piper nigrum L. is crucial for inflorescence specific gene studies for crop improvement. The present study includes comparison of reported CTAB, SDS, and TRIzol methods for extraction of RNA from spikes of black pepper. The quality and yield of RNA was comparatively poor in all these methods. Black pepper spikes, in particular, are rich in polyphenols and secondary metabolites, which comprise a diverse group of naturally occurring compounds contributing to the unique flavour and aroma of the plant. However, the isolation of RNA from black pepper spikes is challenging due to the presence of these compounds, which interfere with the isolation, purification, and resuspension of precipitated RNA, as well as contaminate the RNA pellets through co-precipitation, thus inhibiting downstream applications. Therefore, we have modified original Trizol protocol by inclusion of 2% PVP, 4% β Mercaptoethanol and 1% SDS to reduce the polyphenols at the initial stage of isolation itself. We also increased the incubation time and followed this with repeated chloroform extractions and ethanol washes to remove the remaining contamination. We kept the precipitation at 4ºC to improve the quality and yield of the RNA. The RNA extracted using the modified TRIzol method in the present study was intact, clear, non dispersive with good integrity and high yield (530ng/µl). There was no evidence of DNA or other contaminants in the isolated RNA (A260/A280 – 1.99; A260/A230 – 2.01). The quality was reconfirmed with gel electrophoresis and RT-PCR. Extracted RNA was successfully converted into cDNA and validated by amplification with primer for housekeeping gene β-actin. The present modified TRIzol-based protocol provides valuable insight to isolate high quality RNA from crops with high content of polyphenols, polysaccharides and other metabolites for further downstream techniques.

Keywords

Polyphenols, Secondary metabolites, CTAB, SDS, TRIzol method

Conclusion

To conclude, total RNA extraction from spikes of black pepper using the modified TRIzol method resulted in higher yield and better quality compared to the already reported protocols using CTAB, SDS and TRIzol. The modifications helped in the efficient removal of polyphenols and polysaccharides. The good quality and quantity of the RNA extracted using the present modified method were validated using gel electrophoresis, quantitative nucleic acid detection, and RT-PCR. The modified protocol has the potential for extraction of good quality RNA from samples with high polyphenol and polysaccharide content.

References

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How to cite this article

Manjushri Dinkar Dongare, Swapna Alex, Soni K.B., Sindura K.P., Deepa S. Nair, Roy Stephen and Elizabeth Jose (2023). Modified TRIzol Method for extraction of Good quality RNA from Spikes of Black Pepper (Piper nigrum L.). Biological Forum – An International Journal, 15(10): 228-233.