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Method for extracting total RNA from mature grape fruits

A fruit and grape technology, applied in the field of plant molecular biology, can solve the problems of not being able to extract high-quality total RNA and extraction difficulties, and achieve the effects of good integrity, high sample purity, and economical method

Inactive Publication Date: 2015-11-18
SHANXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

With the extension of storage time, the content of phenolic compounds and secondary metabolites in grape fruit also increases, making RNA extraction more difficult
The traditional CTAB method has been unable to extract high-quality total RNA from grape ripe fruit

Method used

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  • Method for extracting total RNA from mature grape fruits
  • Method for extracting total RNA from mature grape fruits
  • Method for extracting total RNA from mature grape fruits

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Muscat grape fruits in different storage periods of the control group and SO 2 Fresh-keeping group total RNA extraction

[0030] (1) Draw 2ml of the extraction solution in a centrifuge tube and preheat at 65°C; the components of the RNA extraction solution are: 2% (W / V) cetyltrimethylammonium bromide (CTAB), 2% (W / V V) polyvinylpyrrolidone (PVP, K40), 100mmol L -1 Trishydroxymethylaminomethane (Tris-HCl, pH8.0), 100mmol L -1 Ethylenediaminetetraacetic acid (EDTA, pH8.0), 2.5mol·L -1 Sodium chloride (NaCl), 0.5g·L -1 spermidine.

[0031] (2) Take liquid nitrogen quick-frozen control group and SO 2 About 200 mg of Muscat fruit in the fresh-keeping group (storage initial stage, storage 20d and 60d) was ground into powder in liquid nitrogen.

[0032] (3) Add 25 μL of β-mercaptoethanol to the preheated RNA extraction solution in step (1), quickly transfer the grape fruit powder to a centrifuge tube, shake the centrifuge tube to completely volatilize the liquid nitrogen...

Embodiment 2

[0046] Muscat grape fruits in different storage periods of the control group and SO 2 Reverse transcription of total RNA in fresh-keeping group and RT-PCR analysis of proteins related to disease course

[0047] (1) reverse transcription

[0048] cDNA first-strand reaction (product of Beijing Quanshijin Biotechnology Co., Ltd.) system is as follows:

[0049]

[0050] After mixing gently, incubate at 42°C for 30 minutes and heat at 85°C for 5 minutes to inactivate EasyScript RT Enzyme. The synthesized cDNA was stored at -20°C for future use.

[0051] (2) RT-PCR

[0052] The 20μl PCR reaction system is as follows:

[0053]

[0054] X is the amount of cDNA added to each sample when the Actin2 gene is leveled.

[0055] The PCR reaction procedure is:

[0056]

[0057] The annealing temperature (Tm) and cycle number (N) vary according to the amplified gene, and the gene-specific primer sequences are shown in Table 2.

[0058] Table 2 Muscat grape fruits in different s...

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Abstract

The invention provides a method for extracting total RNA from mature grape fruits. The method for extracting the total RNA from the mature grape fruits particularly comprises the steps of extraction by adopting CTAB and chloroform and acidic water phenol purification. According to the method for extracting the total RNA from the mature grape fruits, polyvinylpyrrolidone, spermidine, beta-mercaptoethanol and high-concentration sodium chloride are added into an extracting solution of a traditional CTAB method, the activity of the RNA enzyme can be efficiently restrained, and the interference of polysaccharide and polyphenol substances with RNA can be effectively prevented. In the extraction process, the acidic water phenol purification is utilized for crude extraction of the RNA, DNA which is coprecipitated with the RNA can be removed to the maximum extent, and residual protein and polysaccharides can be cleaned away. The method for extracting the total RNA from the mature grape fruits is low in cost, good in stability, high in purity of extracted RNA samples and good in integrality.

Description

Technical field: [0001] The invention relates to the technical field of plant molecular biology, in particular to a method for extracting total RNA from grape fruit tissue rich in anthocyanins and picked after full maturity. technical background: [0002] RNA extraction is one of the basic techniques of molecular biology. High-quality RNA is a necessary prerequisite for subsequent molecular biology research such as RT-PCR, fluorescent quantitative PCR, Stem-loopRT-PCR, and Northern blot. At present, a variety of RNA extraction methods have been developed, such as guanidine isothiocyanate method, hot boric acid method, sodium dodecyl sulfate (SodiumDodecylSulfate, SDS) method, cetyltrimethylammonium bromide (CetyltrimethylammoniumBromide, CTAB) method, etc. Guanidine isothiocyanate can strongly inhibit the activity of RNase in tissues and extracts. The method is simple to operate, high in yield, and low in cost. However, the purity of the extracted RNA is not very high and f...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 薛美昭仪慧兰
Owner SHANXI UNIV
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