CN104371997A

CN104371997A - Improved method for extracting total RNA in special vegetable tender leaves

Info

Publication number: CN104371997A
Application number: CN201410683649.8A
Authority: CN
Inventors: 张少平; 邱珊莲; 郑开斌; 刘荣章; 林碧珍
Original assignee: Sugarcane Research Institute Fujian Academy Of Agricultural Sciences
Current assignee: Sugarcane Research Institute Fujian Academy Of Agricultural Sciences
Priority date: 2014-11-25
Filing date: 2014-11-25
Publication date: 2015-02-25

Abstract

The invention provides an improved method for extracting total RNA in special vegetable tender leaves. The improved method for extracting the total RNA in the special vegetable tender leaves comprises the following steps: putting an autoclaved and cooled mortar, an autoclave and cooled grinding rod and special vegetable tender leaves into a refrigerator at the temperature of -80 DEG C for more than 4 hours, taking the mortar, the grinding rod and the special vegetable tender leaves out, quickly grinding for about 10s, then sucking right amount of Trizol into the mortar by adopting a pipette, continuously grinding and mixing until jelly is completely dissolved, then sucking the dissolved mixture into a 1.5ml centrifugal tube by adopting a pipette, and then washing, precipitating and carrying out DEPCH2O dissolution, thus the target product, namely the total RNA in special vegetable blades, is obtained. The improved method for extracting the total RNA in the special vegetable tender leaves has the advantages that the problem that RNA is degraded easily (when being placed in the air for a too long time) or a centrifugal tube filled with Trizol mixture bursts (liquid nitrogen is not completely volatilized) as a medicine spoon is used for transferring plant tissue blades smashed by liquid nitrogen into Trizol in the traditional Trizol method for extracting the total RNA in plant tissue blades is solved, the liquid nitrogen is not required to be prepared, and no loss of the ground special vegetable tender leaf powder is produced.

Description

Specialty vegetable tender leaf total serum IgE method is extracted in a kind of improvement

Technical field

The invention belongs to technical field of molecular biology, more specifically relate to a kind of improvement and extract Specialty vegetable tender leaf total serum IgE method.

Background technology

In recent years, along with the development of molecular biotechnology.Become the important foundation of modern molecular biology research based on RNA functional analysis, therefore high quality Total RNAs extraction seems very important for functional gene molecular biology research.In current vegetable material, Total RNAs extraction has the multiple methods such as guanidine isothiocyanate method, phynol method, SDS method, the lithium chloride precipitator method, CTAB method and commercial Trizol RNA isolation kit.And total serum IgE uses Trizol extraction method comparatively general at present, but traditional Trizol method (comprising additive method) is when extracting plant leaf total serum IgE, all need early stage with liquid nitrogen grinding object plant tissue materials, and then with spoon, the plant tissue ground is transferred in Trizol extracting solution.And vegetable material total serum IgE is stable not, in process of the test, grinding smudge cells is to proceed to the operating time in object extracting solution process long, its endogenous and Exogenous RNase all can cause the enzymolysis of object RNA, if and liquid nitrogen grinding object plant tissue materials does not volatilize completely at liquid nitrogen and namely loads Trizol extracting solution, because inequality of being heated easily causes booster in blending process, thus cause human injury.

The present invention is by improving Trizol method extraction step, and the method after improvement can extract high-quality total serum IgE efficiently fast from the tender leaf such as Radix Semiaquilegiae, okra, solves RNA instability, easily degrades, the problems such as the RNA poor quality of extraction.The object RNA that the method is extracted meets reverse transcription, RT-PCR, Northern hybridization and cDNA library completely and builds the biological research needs of equimolecular, for part Specialty vegetable molecular biology research supplying method is used for reference.

Summary of the invention

In order to solve traditional Trizol method extract in the Specialty vegetable such as Radix Semiaquilegiae and okra tender leaf total serum IgE process degrade, booster and need preparation liquid nitrogen and waste more tender leaf material.The invention provides a kind of improvement and extract Specialty vegetable tender leaf total serum IgE method, the method is easy, suitability is strong, takes a firm foundation for obtaining desirable goal gene group total serum IgE.

The Trizol method that the invention provides a kind of improvement extracts Specialty vegetable tender leaf total serum IgE, specifically comprises:

(1) take 0.1-0.6g Specialty vegetable tender leaf, ultrapure water is put into the cooled mortar of autoclaving, is put into-80 DEG C of refrigerator 4-6h together with grinding rod after cleaning and drying;

(2) bring yarn gloves in refrigerator, take out the mortar that Specialty vegetable tender leaf is housed, tender leaf 10-12s is ground fast with grinding rod, (tender leaf mass unit g with Trizol volume unit ml ratio is: 0.1:1), continues grinding jelly to dissolving completely in mortar to draw appropriate Trizol with liquid-transfering gun again;

(3) be dissolved with the Trizol mixture of object RNA in 1.5ml centrifuge tube with liquid-transfering gun from absorption in mortar, often pipe dress about 1ml, room temperature leaves standstill 2min;

(4) often pipe adds 0.2ml chloroform, and leave standstill 2min after vibration mixing, 4 DEG C of centrifugal 10min of 12000g, get supernatant;

(5) add the Virahol that 0.5ml is ice-cold, mix gently, leave standstill 10min, 4 DEG C of centrifugal 10min of 12000g, abandon supernatant;

(6) 1ml DEPC H is added ₂75% ethanol of O process, washing precipitation, 4 DEG C, the centrifugal 5min of 8000g, abandons supernatant, adds appropriate DEPC H after super clean bench dries ₂o dissolves.

In step (2), Specialty vegetable tender leaf quality (unit g) and Trizol volume (units/ml) are than being: 0.1:1.

The method is that cooled for autoclaving mortar, grinding rod and object Specialty vegetable tender leaf are together put into-80 DEG C of more than refrigerator 4h, about 10s is ground in rear taking-up fast, appropriate Trizol is drawn in mortar again with liquid-transfering gun, continue grinding Specialty vegetable tender leaf jelly to dissolving completely, be drawn in 1.5ml centrifuge tube with liquid-transfering gun again, after through washing, precipitation and DEPC H ₂o dissolves, and namely obtains object Specialty vegetable tender leaf total serum IgE.When present method solves traditional Trizol method extraction plant tissue blade total serum IgE, with spoon, the plant tissue blade that liquid nitrogen grinds is proceeded in Trizol process, RNA is easily caused to degrade (in air, storage period is long), or the centrifuge tube explosion (liquid nitrogen does not vapor away completely) of Trizol mixture is housed, the method is without the need to preparing liquid nitrogen and not losing the Specialty vegetable tender leaf powder ground simultaneously.

Accompanying drawing explanation

Fig. 1 each sample extracts RNA electrophorogram, and wherein M is Maker, 1 be Radix Semiaquilegiae tender leaf, 2 be okra tender leaf, 3 for Gynura divaricata tender leaf and 4 be balsam pear tender leaf, 28S, 18S and 5S represent 28S RNA, 18S RNA, 5S RNA band.

embodiment

Embodiment 1

(1) take 0.1g Specialty vegetable tender leaf, ultrapure water is put into the cooled mortar of autoclaving, is put into-80 DEG C of refrigerator 4h together with grinding rod after cleaning and drying;

(2) bringing yarn gloves in refrigerator, take out the mortar that Specialty vegetable tender leaf is housed, grind tender leaf 10s fast with grinding rod, then draw 1mlTrizol in mortar with liquid-transfering gun, continuing grinding jelly to dissolving completely;

(3) be dissolved with in Trizol mixture to a 2 1.5ml centrifuge tube of object RNA with liquid-transfering gun each 0.5ml that draws in mortar, room temperature leaves standstill 2min;

(6) 1ml DEPC H is added ₂75% ethanol of O process, washing precipitation, 4 DEG C, the centrifugal 5min of 8000g, abandons supernatant, and super clean bench dries rear often pipe and adds 50ulDEPC H ₂o dissolves.A small amount of RNA that this method is extracted can be further used for the reaction such as reverse transcription and RT-PCR.

Embodiment 2

(1) take 0.3g Specialty vegetable tender leaf, ultrapure water is put into the cooled mortar of autoclaving, is put into-80 DEG C of refrigerator 5h together with grinding rod after cleaning and drying;

(2) bringing yarn gloves in refrigerator, take out the mortar that Specialty vegetable tender leaf is housed, grind tender leaf 12s fast with grinding rod, then draw 3mlTrizol in mortar with liquid-transfering gun, continuing grinding jelly to dissolving completely;

(3) be dissolved with in Trizol mixture to a 3 1.5ml centrifuge tube of object RNA with liquid-transfering gun each 1ml that draws in mortar, room temperature leaves standstill 2min;

(6) 1ml DEPC H is added ₂75% ethanol of O process, washing precipitation, 4 DEG C, the centrifugal 5min of 8000g, abandons supernatant, and super clean bench dries rear often pipe and adds 100ulDEPC H ₂o dissolves.The middle amount RNA that this method is extracted can be further used for the reactions such as Northern hybridization.

Embodiment 3

(1) take 0.6g Specialty vegetable tender leaf, ultrapure water is put into the cooled mortar of autoclaving, is put into-80 DEG C of refrigerator 6h together with grinding rod after cleaning and drying;

(2) bringing yarn gloves in refrigerator, take out the mortar that Specialty vegetable tender leaf is housed, grind tender leaf 15s fast with grinding rod, then draw 6mlTrizol in mortar with liquid-transfering gun, continuing grinding jelly to dissolving completely;

(3) be dissolved with in Trizol mixture to a 6 1.5ml centrifuge tube of object RNA with liquid-transfering gun each 1ml that draws in mortar, room temperature leaves standstill 2min;

(6) 1ml DEPC H is added ₂75% ethanol of O process, washing precipitation, 4 DEG C, the centrifugal 5min of 8000g, abandons supernatant, and super clean bench dries rear often pipe and adds 100ulDEPC H ₂o dissolves.The relatively large RNA that this method is extracted can be further used for the reactions such as cDNA library structure.

Embodiment 4

By aforesaid method, respectively take 0.2g Radix Semiaquilegiae, okra, Gynura divaricata and balsam pear tender leaf, carry out its RNA extraction, the RNA that obtains respectively gets 1ul, use 1% agarose, 200V electrophoresis 12 min, dye 2min in ethidium bromide, take a picture with gel imaging instrument and detect (see figure 1), result obtains good quality and higher yield, and the RNA sample carried is all very complete, 28S, 18S and 5S RNA is all high-visible, and electrophoresis band brightness also reflects that obtaining RNA has higher concentration.

Claims

1. Specialty vegetable tender leaf total serum IgE method is extracted in an improvement, it is characterized in that: the method is that cooled for autoclaving mortar, grinding rod and object Specialty vegetable tender leaf are together put into-80 DEG C of refrigerator 4-6h, rear taking-up is ground fast, appropriate Trizol is drawn in mortar again with liquid-transfering gun, continue grinding Specialty vegetable tender leaf jelly to dissolving completely, be drawn in 1.5ml centrifuge tube with liquid-transfering gun again, after through washing, precipitation and DEPC H ₂o dissolves, and namely obtains object Specialty vegetable tender leaf total serum IgE.

2. Specialty vegetable tender leaf total serum IgE method is extracted in one improvement according to claim 1, it is characterized in that: described method concrete steps are: (1) takes 0.1-0.6g Specialty vegetable tender leaf, after ultrapure water is cleaned and is dried, put into the cooled mortar of autoclaving, put into-80 DEG C of refrigerator 4-6h together with grinding rod;

(2) bring yarn gloves in refrigerator, take out the mortar that Specialty vegetable tender leaf is housed, grind tender leaf 10-12s fast with grinding rod, then draw in appropriate Trizol to mortar with liquid-transfering gun and continue grinding jelly to dissolving completely;

(3) be dissolved with the Trizol mixture of object RNA in 1.5ml centrifuge tube with liquid-transfering gun from absorption in mortar, often pipe dress 1ml, room temperature leaves standstill 2min;

(6) 1ml DEPC H is added ₂75% ethanol of O process, washing precipitation, 4 DEG C, the centrifugal 5min of 8000g, abandons supernatant, adds the DEPC H of 0.1 ml 1% after super clean bench dries ₂o dissolves.

3. Specialty vegetable tender leaf total serum IgE method is extracted in one improvement according to claim 1, it is characterized in that: in step (2), Specialty vegetable tender leaf quality (unit g) and Trizol volume (units/ml) are than being: 0.1:1.