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Extracting method for total RNA (Ribonucleic Acid) of galangal

An extraction method and technology of galangal, applied in the biological field, can solve problems such as difficulty in extracting RNA from galangal tissue, loss of activity, etc., and achieve the effects of saving extraction time, simple operation and high yield

Inactive Publication Date: 2014-04-23
SOUTH CHINA NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The complex diversity of secondary metabolites in galanga tissue, especially the rich content of polyhydroxy flavonols, flavonols are easily oxidized, and their oxidation products can cause RNA degradation and loss of activity or form insoluble complexes. High content of flavonols can lead to difficulty in RNA extraction from galanga tissue
[0005] At present, there is no literature or patent related to Galanga RNA extraction and subsequent molecular research

Method used

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  • Extracting method for total RNA (Ribonucleic Acid) of galangal
  • Extracting method for total RNA (Ribonucleic Acid) of galangal
  • Extracting method for total RNA (Ribonucleic Acid) of galangal

Examples

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Embodiment 1

[0032] Extraction of total RNA from fresh young leaves of galanga, comprising the following steps:

[0033] (1) Take 300 mg of young galangal leaves, put them into a pre-cooled mortar, add 1.5 mL of pre-cooled lysate extract, 500 μL of pre-cooled acetone into the mortar, add liquid nitrogen repeatedly to grind into powder, and place at room temperature for 40 minutes , transfer the ground homogenate to a 2mL centrifuge tube, centrifuge at 12000g at 4°C for 15min, transfer the colorless supernatant layer to another new centrifuge tube to obtain solution A; the lysed extract consists of the following components : 5mol / L Guanidine Isothiocyanate, 25mmol / L Sodium Citrate, 0.5% (w / v) Sodium Lauryl Sarcosinate, 4% (w / v) Soluble Polyvinylpyrrolidone (PVP, K40), 4% (v / v) β-mercaptoethanol;

[0034] (2) Add 1 / 3 volume of Tris saturated phenol (pH 8.8) and 1 / 3 volume of 5mol / L KAc solution to the solution A obtained in step (1), mix thoroughly, and place at room temperature for 15-30mi...

Embodiment 2

[0043] Extraction of total RNA from liquid nitrogen quick-frozen galangal leaves, comprising the following steps:

[0044] (1) After 200mg of young galanga leaves are quick-frozen in liquid nitrogen, put them into a pre-cooled mortar, add 1.0mL of pre-cooled lysate extract and 500μL of pre-cooled acetone to the mortar, add liquid nitrogen repeatedly and grind them into powder , placed at room temperature for 30min, transferred the homogenate to a 2mL centrifuge tube, centrifuged at 12000g at 4°C for 15min, transferred the colorless supernatant layer to another new centrifuge tube to obtain solution A;

[0045] (2) Add 1 / 3 volume of Tris saturated phenol (pH 8.8) and 1 / 3 volume of 5mol / L KAc solution to the solution A obtained in step (1), mix thoroughly, and place at room temperature for 15-30min, 4 Centrifuge at 12000g for 15min, transfer the aqueous phase to a new centrifuge tube to obtain solution B;

[0046] (3) Add an equal volume of chloroform to the solution B obtained...

Embodiment 3

[0051] The cDNA reverse transcription of galanga total RNA and the amplification of internal reference gene 18S rRNA gene comprise the following steps:

[0052] (1) Prepare the following template RNA in a PCR tube: Add 2 μL total RNA from galanga quick-frozen leaves (1.24 μg) or fresh leaves (1.78 μg), 1 μL Oligo (dT) 15 primer (0.5 μg), 7 μL DEPC-ddH 2 O, after mixing, keep the temperature at 70°C for 10 minutes, place on ice for 2 minutes, and centrifuge for a few seconds to make the solution aggregate at the bottom of the tube;

[0053] (2) Add the following reaction solution to the PCR tube in step (1): 2μL M-MLV5×Reaction Buffer, 0.5μL dNTP (10mM each), 0.25μL Ribolock RNase Inhibitor, 0.5μL M-MLV RT, 1.75μL DEPC-ddH 2 P, after mixing, react at 42°C for 1.5h, take it out and heat at 70°C for 5min to inactivate reverse transcriptase, and obtain cDNA after cooling on ice;

[0054] (3) Take 1 μL of fresh or liquid nitrogen quick-frozen galanga young leaves and reverse tran...

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Abstract

The invention discloses an extracting method for the total RNA (Ribonucleic Acid) of galangal. The method is improved on various aspects on the basis of the conventional guanidine thiocyanate-phenol-chloroform extraction method according to the characteristic of rich secondary metabolism product of the galangal and particularly rich polyhydric flavonol, i.e., acetone, water-soluble polyvinylpyrrolidone (PVP) and beta-mercaptoethanol are added in an RNA extracting solution. The acetone can be used for dissolving colored matters such as flavonols and the like; the water-soluble PVP can be sufficiently combined with phenol substances to form chelates; the beta-mercaptoethanol as a strong reducing agent can be used for providing a reducing condition so that polyphenol substances are not easy to oxidize; the three substances synergistically take effects, so that the interference and obstruction of the polyphenol substances are effectively inhibited. According to the invention, a crudely-extruded sediment of the RNA is treated by using Tris saturated phenol and high-concentration potassium acetate after being obtained, and part of proteins and polysaccharides can be simultaneously removed.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for extracting total RNA of medicinal plants, in particular to a method for extracting total RNA of galanga. Background technique [0002] The extraction of RNA with high purity and integrity from plant tissues is the necessary prerequisite and key for cDNA reverse transcription, in vitro translation, cDNA library establishment, RT-PCR and differential analysis in the study of plant gene function and expression regulation. In practice, it is often found that even if different tissues of the same plant have different RNA extraction methods; even the same tissue material of the same plant, but from different genotype plants, the RNA extraction methods may also be different. . Therefore, for a certain plant or its tissue, its corresponding RNA extraction method must be explored and practiced before it can be established. [0003] Galangal (Alpinia officinarum Hanc...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 李淑彬徐诗如周仁超
Owner SOUTH CHINA NORMAL UNIVERSITY
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