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High-quality RNA isolation from pigment-rich Dendrobium flowers

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Abstract

Isolation of high-quality RNA from Dendrobium flowers is challenging because of the high levels of pigment, polysaccharides, and polyphenols. In the present study, an efficient CTAB method for RNA extraction from the pigment-rich flowers of Dendrobium was optimised. The optimised method yielded high quantities of RNA (10.1–12.9 µg/g). Spectrophotometric values of A260/280 in the range of 2.2 to 2.4 and A260/230 values of 2.0 suggested that the isolated RNA was free of polyphenols, polysaccharides, and protein contaminants. RNA integrity numbers determined by microfluidics were in the range of 7.9–8.9 indicative of intact RNA. In the improved method, the addition of 3 M NaCl and 3% PVP-10 in the extraction buffer, followed by an incubation period of 45 min at 65 °C, eliminated most of the polysaccharides, polyphenolic compounds, and denatured protein. Extraction with phenol:chloroform:isoamyl alcohol (125:24:1) effectively removed pigments from the aqueous phase, while the precipitation of RNA with lithium chloride minimised the co-precipitation of protein, DNA, and polysaccharide and resulted in the extraction of high quality of RNA. The suitability of the RNA for downstream processing was confirmed via RT-PCR amplification of Chalcone synthase gene from cDNA prepared from RNA isolated from different developmental stages of the flower of a Dendrobium hybrid. The present method will be highly useful for the isolation of RNA from pigment, polyphenol, and polysaccharide-rich plant tissues.

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Abbreviations

bp:

Base pair

cDNA:

Complimentary DNA

CHS:

Chalcone synthase

C:I:

Chloroform:isoamyl alcohol

P:C:I:

Phenol:chloroform:isoamyl alcohol

CTAB:

Cetyltrimethylammonium bromide

°C:

Degrees Celsius

cm:

Centimetre

DEPC:

Diethyl pyrocarbonate

DNase:

Deoxyribonuclease

EDTA:

Ethylenediaminetetraacetic acid

EtBr:

Ethidium bromide

FW:

Fresh weight

LiCl:

Lithium chloride

M:

Molarity

ml:

Millilitre

mM:

Millimolar

min:

Minutes

NaCl:

Sodium chloride

psi:

Pound-force per square inch

PVP-10:

Polyvinylpyrrolidone-10

RNase:

Ribonuclease

rRNA:

Ribosomal RNA

RT-PCR:

Reverse transcription polymerase chain reaction

s:

Second

SDS:

Sodium dodecyl sulfate

TBE:

Tris-borate-EDTA

Tris–HCl:

Tris hydrochloride

µl:

Microlitre

ng:

Nanogram

w/v:

Weight per volume

v/v:

Volume per volume

%:

Percentage

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Acknowledgements

This work was supported by the Frontier Research Grant (FRG 2017, FG026-17AFR) and CEBAR Research University Grants (RU006-2017 and RU006-2018).

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Authors and Affiliations

  1. Centre for Research in Biotechnology for Agriculture, University of Malaya, Kuala Lumpur, 50603, Malaysia

    Muhammad-Asyraf Khairul-Anuar, Purabi Mazumdar, Su-Ee Lau, Tian Tian Tan & Jennifer Ann Harikrishna

  2. Faculty of Science, University of Malaya, Kuala Lumpur, 50603, Malaysia

    Jennifer Ann Harikrishna

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  1. Muhammad-Asyraf Khairul-Anuar
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  2. Purabi Mazumdar
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  3. Su-Ee Lau
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  4. Tian Tian Tan
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Contributions

MAKA, PM, S-EL, and TTT conducted experimental work; PM and JAH made the experimental design. All authors contributed to manuscript preparation and to editing of both original and revised manuscript.

Corresponding author

Correspondence to Jennifer Ann Harikrishna.

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The authors declare that they have no conflicts of interest.

Electronic supplementary material

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Supplementary material 1 (DOCX 39 kb)

13205_2019_1898_MOESM2_ESM.pptx

Supplementary Fig. S1 Flow diagram of cDNA synthesis from total RNA (SuperScript® III First-Strand Synthesis system (Invitrogen, USA)

13205_2019_1898_MOESM3_ESM.pptx

Supplementary Fig. S2 Chalcone synthase (CHS) gene sequence of Dendrobium hybrid cultivar Sonia Earsakul (KC345011.1) with primer binding sites (Orange letter denotes introns and blue- bold letter denotes primer binding sites)

13205_2019_1898_MOESM4_ESM.pptx

Supplementary Fig. S3 Aqueous fraction collected after P:C:I steps of RNA isolation method. M1: RNA isolation method of Kiefer et al. 2000, M2: RNA isolation method of Su et al. 2011, M3: RNA isolation method of Gao et al. 2016, M4: RNA isolation method of Liu et al. 2018, and M5: improved CTAB method

13205_2019_1898_MOESM5_ESM.pptx

Supplementary Fig. S4 RNA pellet obtained from the mature flower of Dendrobium hybrid. M1: RNA isolation method of Kiefer et al. 2000, M2: RNA isolation method of Su et al. 2011, M3: RNA isolation method of Gao et al. 2016, M4: RNA isolation method of Liu et al. 2018, and M5: improved CTAB method

13205_2019_1898_MOESM6_ESM.pptx

Supplementary Fig. S5 Agarose gel electrophoresis of total RNA isolated from the mature flower of four Dendrobium hybrid flowers. Lanes 1–3: Dendrobium Burmese ruby × Dendrobium Mae-klong River; Lanes 4–6: Dendrobium Burana Jade × [Dendrobium Bertha Chong × Dendrobium Imelda Romualdez]), Lanes 7–9: Dendrobium Trudy Brandt × Dendrobium Udom Blue Angel, Lanes 10–12: Dendrobium Aridang × Dendrobium Burana Sundae and Lane 13: 1 kb DNA Ladder (Promega, USA)

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Khairul-Anuar, MA., Mazumdar, P., Lau, SE. et al. High-quality RNA isolation from pigment-rich Dendrobium flowers. 3 Biotech 9, 371 (2019). https://doi.org/10.1007/s13205-019-1898-y

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