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Rapid and Efficient Isolation of High Quality Nucleic Acids from Plant Tissues Rich in Polyphenols and Polysaccharides

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Abstract

Isolation of high quality nucleic acids from plant tissues rich in polysaccharides and polyphenols is often difficult. The presence of these substances can affect the quality and/or quantity of the nucleic acids isolated. Here, we describe a rapid and efficient nucleic acids extraction protocol that in contrast to other methods tested, effectively purify high quality nucleic acids from plant tissues rich in polysaccharides and polyphenolic compounds such as different grape tissues and fruit tissue of fruit trees. The nucleic acids isolated with this protocol were successfully used for many functional genomic based experiments including polymerase chain reaction, reverse transcription polymerase chain reaction (RT-PCR), cloning, and semiquantitative RT-PCR.

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Notes

  1. Pestles, mortars and all glassware used in the isolation of total RNA from plant material are baked for 2 h at 180ºC. Also, 2 ml Eppendorf tubes and tips are immersed overnight in 0.1% DEPC treated water and then autoclaved for 30 min.

  2. For isolation of total RNA, EDTA, NaCl, NaOAc, and LiCl are treated with 0.1% DEPC solution and stored overnight, then are autoclaved for 30 min. Tris–HCl and 70% EtOH are prepared with 0.1% DEPC-treated, autoclaved distilled water.

  3. Isolation of total RNA should carry out at 4°C, whereas genomic DNA extraction is performed at room temperature.

  4. The extraction procedure can be scaled up or down depending on the amount of initial plant tissue available and the quantity of nucleic acid required.

  5. In the isolation of total RNA, tissues should grind to a fine powder, but for genomic DNA extraction, it is not necessary that tissues grind completely.

  6. At this point, the pellet is white and clear. Do not let the pellet dry completely. Immediately resuspend the pellet in an adequate volume of TE buffer or DEPC-treated water.

  7. If the pellet is unclear, centrifuge at 14000 rpm for 1 min to remove any trace of plant material in nucleic acid solution.

  8. At this point, it is important to remove all supernatant to prevent carryover of contaminating substances.

Abbreviations

Chl:Iaa:

Chloroform-isoamylalcohol

CTAB:

Cetyltrimethylammonium bromide

DEPC:

Diethyl pyrocarbonate

EDTA:

Ethylenediaminetetra acetic acid

EtBr:

Ethidium bromide

EtOH:

Ethanol

FW:

Fresh weight

PVPP:

Polyvinylpolypyrrolidone

PVP:

Polyvinylpyrrolidone

NaOAc:

Sodium acetate

RT-PCR:

Reverse transcription polymerase chain reaction

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Correspondence to Raheem Haddad.

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Japelaghi, R.H., Haddad, R. & Garoosi, GA. Rapid and Efficient Isolation of High Quality Nucleic Acids from Plant Tissues Rich in Polyphenols and Polysaccharides. Mol Biotechnol 49, 129–137 (2011). https://doi.org/10.1007/s12033-011-9384-8

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