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A simple and efficient protocol for isolation of high quality functional RNA from different tissues of turmeric (Curcuma longa L.)

Physiology and Molecular Biology of Plants volume 20, pages 263–271 (2014)Cite this article

Abstract

Many experiments in plant molecular biology require processing of a large number of RNA samples and in some cases large quantities are required for a single application. In turmeric, a major spice and medicinal plant, a protocol for RNA isolation is not available. The major difficulty encountered while using other popular protocols is the low yield and quality of RNA which hampers the downstream applications like qRT-PCR, cDNA synthesis and micro RNA isolation. Commercial kits though available are costly and were found to be unsuccessful in case of rhizomes and root tissues that are rich in polyphenols, polysaccharides and alkaloids. It was thus felt that a quick, handy and cheap protocol of total RNA isolation from different tissues of turmeric was required for day to day working in our lab. The new protocol utilizes SDS based extraction buffer including β-mercaptoethanol and PVP with sequential acid phenol:chloroform extraction to remove polyphenols and proteins, followed by the purification with sodium acetate to eliminate polysaccharides. The protocol is simple and can be completed in less than 3 h. The RNA yield from rhizome was higher by more than fivefold with both A260/280 and A260/230 ratio in the range of 1.8–2.0. The protocol worked well with leaf, rhizome, pseudostem and root tissues with RIN >7.0 and the isolated RNA could be successfully used for cDNA synthesis, RT-PCR, qRT-PCR and small RNA isolation including microRNA.

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Abbreviations

DEPC:

Diethylpyrocarbonate

RT-PCR:

Reverse transcription-polymerase chain reaction

PVP:

Polyvinylpyrrolidone

SDS:

Sodium dodecyl sulphate

EDTA:

Ethylene diamine tetraacetic acid

NaOAc:

Sodium acetate

PEG:

Polyethylene glycol

RIN:

RNA Integrity Number

qRT-PCR:

Quantitative real time polymerase chain reaction

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Acknowledgments

We thank Director, IISR for providing necessary facilities to carry out this work.

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Affiliations

  1. Division of Crop Improvement and Biotechnology, Indian Institute of Spices Research, Calicut, 673 012, Kerala, India

    K. Deepa, T. E. Sheeja, R. Santhi, B. Sasikumar, Anu Cyriac, P. V. Deepesh & D. Prasath

Authors
  1. K. Deepa
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  2. T. E. Sheeja
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  3. R. Santhi
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  4. B. Sasikumar
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  5. Anu Cyriac
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Correspondence to T. E. Sheeja.

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Deepa, K., Sheeja, T.E., Santhi, R. et al. A simple and efficient protocol for isolation of high quality functional RNA from different tissues of turmeric (Curcuma longa L.). Physiol Mol Biol Plants 20, 263–271 (2014). https://doi.org/10.1007/s12298-013-0218-y

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  • DOI: https://doi.org/10.1007/s12298-013-0218-y

Keywords

  • Curcumin
  • Polysaccharides
  • Nucleic acid isolation
  • RIN
  • sRNA
  • RT-PCR
  • qRT-PCR