Abstract
Isolation of high-quality RNA from coffee is challenging because of high level of polysaccharides, polyphenols and other secondary metabolites. In the present study, a rapid and efficient RNA extraction protocol from different tissues of coffee was optimized. Sufficiently high quality and quantity (225.6–454.8 µg/g) of RNA was obtained by using the optimized protocol. The presence of two distinct bands of 28S rRNA and 18S rRNA in agarose gel proved the intactness of the RNA samples. The average spectrophotometric values of the isolated RNA ranged from 1.96 to 2.02 (A260/280) and 1.95 to 2.14 (A260/230), indicating the high quality of RNA devoid of polyphenols, polysaccharides and protein contamination. In the optimized protocol, addition of PVPP to the extraction buffer and a brief incubation of samples at 65 °C and subsequent purification with potassium acetate resulted in good-quality RNA isolation. The suitability of RNA for downstream processing was confirmed by PCR amplification with cytochrome c oxidase gene-specific primers. The amplification of a single 392 bp fragment using cDNA and 1.5 kb fragment using genomic DNA samples confirmed the absence of DNA contamination. The present protocol is rapid and yielded good quality and quantity of RNA suitable for functional genomics studies.
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Acknowledgements
The authors thank Dr. Y. Raghuramulu, Director of Research, Central Coffee Research Institute, India, for providing laboratory facilities and encouragement. Funding support from Coffee Board, Govt. of India, is gratefully acknowledged.
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The corresponding author designed the experiment and wrote the manuscript. The first and second authors carried out the experiments and participated in manuscript preparation. All the authors read and approved the manuscript.
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Huded, A.K.C., Jingade, P. & Mishra, M.K. A rapid and efficient SDS-based RNA isolation protocol from different tissues of coffee. 3 Biotech 8, 183 (2018). https://doi.org/10.1007/s13205-018-1209-z
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DOI: https://doi.org/10.1007/s13205-018-1209-z