CN104313014A - Method for rapidly extracting total ribonucleic acid (RNA) of fir - Google Patents

Abstract

The invention relates to a method for rapidly extracting total ribonucleic acid (RNA) from plant tissues, and particularly relates to the method for extracting total RNA from the plant tissues rich in secondary metabolites like polyphenol. The invention aims to providing the simple, economic and efficient method for rapidly extracting total RNA of a fir, which offers more choices for researchers in work. Due to adoption of the method, the extracted RNA has high quality, can meet the requirements of gene isolation and expression analysis like reverse transcription-polymerase chain reaction (RT-PCR), Northern hybridization and so on, and can greatly lower the cost of experiments. The method can rapidly extract the RNA of the fir tissue through mixing extraction of sodium dodecyl sulfate (SDS) extracting solution and mercaptoethanol as well as purification by a centrifuging column, so that the time is greatly shortened, the cost of the experiment is lowered, and the extracted RNA has high purity and can meet the requirement of molecular biology study.

Description

A kind of method of rapid extraction China fir total serum IgE

Technical field

The present invention relates to a kind of method of rapid extraction total serum IgE from plant tissue, particularly a kind of method extracting total serum IgE from the plant tissue being rich in the secondary metabolites such as polyphenol.

Background technology

China fir ( cunninghamia lanceolata(Lamb.) Hook.) be the important yielding timber plantationsion of south China.In recent years, along with the development of Protocols in Molecular Biology, also start gradually both at home and abroad to utilize molecular biology method to carry out the correlative study of China fir, and extract from China fir tissue high-quality total serum IgE be carry out these research basis and key.

China fir is rich in the secondary metabolites such as polyphenol, polyphenol is easily oxidized to the quinones substance of brown, RNA can be made to degrade, RNase and polyphenoloxidase all belong to protein, obtain complete, high-quality total serum IgE and just must can remove albumen, secondary metabolite is then easy to RNA and combines, and hinders the separation of RNA.Therefore, high-quality China fir total serum IgE be obtained, just must remove polyphenol and secondary metabolite, suppress the oxidation of polyphenol.

At present, the extracting method of frequently seen plants material total serum IgE is more, mainly contains CTAB method, modified CTAB method, Trizol method, guanidine isothiocyanate method, hot borate method etc., but not yet has a kind of method can extracting total serum IgE fast from China fir tissue.We are with low cost in the urgent need to one, simple, convenient, efficient extracting method.

Summary of the invention

The object of the present invention is to provide a kind of method that is simple, economic, rapid extraction China fir total serum IgE efficiently, for researchist provides more selection in real work.The RNA quality extracted is high, can meet the needs of the researchs such as gene isolation and expression analysis such as RT-PCR, Northern hybridization, greatly can reduce the cost of experiment simultaneously.

The method of a kind of rapid extraction China fir total serum IgE provided by the present invention, comprises the following steps:

(1) in 1.5 ml centrifuge tubes, following liquid is mixed: 1 ml SDS extracting solution and 40-60 μ l mercaptoethanol, be designated as extracting solution A;

(2) get the fresh China fir material of 90-120mg to be put in 2 ml centrifuge tubes, and add steel ball, be put into rapidly quick-frozen in liquid nitrogen, use high-throughput tissue grinder grind into powder;

(3) in the centrifuge tube after grinding, add rapidly 0.8-1.5 ml extracting solution A, vortex mixes, and room temperature leaves standstill 8-15 min, 4 DEG C, the centrifugal 10-20 min of 13000g;

(4) draw in 1.5 new ml centrifuge tubes of 500-700 μ l supernatant to, add 200-400 μ l 5 mol/L NaCl and 400-600 μ l chloroform, mixing, 4 DEG C, the centrifugal 4-7 min of 13000g;

(5) draw 500-700 μ l supernatant, add isopyknic water-saturated phenol: the mixed solution of chloroform v/v=1:1, mixing, 4 DEG C, the centrifugal 4-7 min of 13000g;

(6) draw 300-500 μ l supernatant in new 1.5 ml centrifuge tubes, add the Virahol of equal-volume precooling, under room temperature, on side-sway shaking table, shake up 10 min;

(7) liquid after above-mentioned shaking up is drawn in centrifugal column, 4 DEG C, the centrifugal 1-3 min of 13000g;

(8) the washing with alcohol precipitation of 75% precooling is added, 4 DEG C, the centrifugal 1-3 min of 13000g;

(9) repeating step (8);

(10) liquid is abandoned, 4 DEG C, the empty centrifugal 1-3 min of 13000g;

(11) collection tube changing bottom be new 1.5 ml without RNA enzyme centrifuge tube, add 30-50 μ l DEPC water to above-mentioned centrifugal column central authorities, leave standstill 2-5 min, 4 DEG C, the centrifugal 1-3 min of 13000g;

(12) liquid bottom centrifugal rear centrifuge tube is sucked in centrifugal column again, leave standstill 2 ~ 5 min, 4 DEG C, the centrifugal 1-3 min of 13000g;

(13) abandon centrifugal column, new collection tube-20 DEG C is saved backup.

SDS extracting solution configures: 0.25M NaCl, 0.05M Tris-HCl, pH=7.5,20mM EDTA, pH=8.0,1%SDS.

More preferably, a kind of method of rapid extraction China fir total serum IgE, comprises the following steps:

(1) in 1.5 ml centrifuge tubes, following liquid is mixed: 1 mlSDS extracting solution and 50 μ l mercaptoethanols, be designated as extracting solution A;

(2) get the fresh China fir material of 100mg to be put in 2 ml centrifuge tubes, and add 25 mm steel balls, be put into rapidly quick-frozen in liquid nitrogen, use high-throughput tissue grinder grind into powder;

(3) in the centrifuge tube after grinding, add rapidly 1 ml extracting solution A, vortex mixes, and room temperature leaves standstill 10 min, 4 DEG C, centrifugal 15 min of 13000g;

(4) draw in 1.5 new ml centrifuge tubes of 600 μ l supernatants to, add 300 μ l 5 mol/L NaCl and 500 μ l chloroforms, mixing, 4 DEG C, centrifugal 5 min of 13000g;

(5) draw 600 μ l supernatants, add isopyknic water-saturated phenol: the mixed solution of chloroform v/v=1:1, mixing, 4 DEG C, centrifugal 5 min of 13000g;

(6) draw 400 μ l supernatants in new 1.5 ml centrifuge tubes, add the Virahol of equal-volume precooling, under room temperature, on side-sway shaking table, shake up 10 min;

(7) liquid after above-mentioned shaking up is drawn in centrifugal column, 4 DEG C, centrifugal 1 min of 13000g;

(8) the washing with alcohol precipitation of 500 μ l 75% precoolings is added, 4 DEG C, centrifugal 1 min of 13000g;

(9) repeating step (8);

(10) liquid is abandoned, 4 DEG C, empty centrifugal 1 min of 13000g;

(11) collection tube changing bottom be new 1.5 ml without RNA enzyme centrifuge tube, add 50 μ l DEPC water to above-mentioned centrifugal column central authorities, leave standstill 2 min, 4 DEG C, centrifugal 1 min of 13000g;

(12) liquid bottom centrifugal rear centrifuge tube is sucked in centrifugal column again, leave standstill 2 min, 4 DEG C, centrifugal 1 min of 13000g;

(13) abandon centrifugal column, new collection tube-20 DEG C is saved backup.

This invention advantage is, can carry out rapid extraction to the RNA of China fir tissue, greatly shortens the time and reduces experimental cost.Such as, 1ml extracting solution A effectively can extract the total serum IgE (accompanying drawing 1) of 100mg Chinese Fir Leaves and root, OD _260/280between 1.9-2.1, the RNA purity of extraction is higher, can meet the demand of molecular biology research; Extract the total serum IgE of China fir tissue by traditional CTAB method or modified CTAB method, step is more loaded down with trivial details, longer (overnight precipitation) consuming time; Also needing the step removing DNA and just can obtain the high RNA of quality comparation, also because adding this step, causing the comparision contents of the RNA finally obtained low.

Accompanying drawing explanation

Fig. 1 is that agarose gel electrophoresis detects China fir total tissue RNA, the design sketch of gained of taking pictures with gel imaging system after electrophoresis: a is well, and b is 28S RNA, c be 18S RNA, d is 5S RNA; A, B are Chinese Fir Leaves, and C, D are Root-bark of Chinese Fir.

Fig. 2 is China fir total serum IgE electrophorogram (CTAB method: M is marker, A is Lignum seu Ramulus Cunninghamiae Lanceolatae leaf, and B is Root-bark of Chinese Fir).

Embodiment

The experimental technique used in following embodiment if no special instructions, is ordinary method.

Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.

Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in this:

Embodiment 1

1, the preparation of solution

(1) 0.1%(v/v) DEPC water: add 1 ml coke diethyl phthalate (DEPC) in 1000 ml ultrapure waters, on magnetic stirring apparatus, stir more than 4h, 121 DEG C of autoclaving 20 min, shake up, for subsequent use after cooling, all solution below used all need with 0.1%(v/v) preparation of DEPC water.

(2) 1M Tris-HCl(pH=7.5) 50 ml: take 12.11 g Tris alkali and be dissolved in about 80 ml DEPC water, regulate pH to 7.5 with concentrated hydrochloric acid, be settled to 100 ml.

(3) 5M NaCl 100 ml: take 29.22 g NaCl and be dissolved in about 80 ml DEPC water, be settled to 100 ml.

(4) 0.5M EDTA(pH=8.0) 100 ml: take 18.61 g Na ₂eDTA2H ₂o is dissolved in 80 ml DEPC water, heating for dissolving, regulates pH to 8.0 with NaOH.

(5) SDS extracting solution: 0.25M NaCl, 0.05M Tris-HCl(pH=7.5), 20mM EDTA, 1%(M/V) SDS.Take 1g SDS to be dissolved in 20 ml DEPC water, then add 5 ml 5M NaCl, 5ml 1M Tris-HCl, 4 ml 0.5M EDTA, are settled to 100 ml with DEPC water.Because beta-mercaptoethanol is easily degraded, now add before using (extracting solution A:1ml SDS extracting solution and the mixing of 40-60 μ l beta-mercaptoethanol vortex).

2, experimental article process

Glassware in leaching process toasts 6h continuously at 180 DEG C; Plastic centrifuge tube and the equal 121 DEG C of autoclaving 20min of rifle head, then 80 DEG C of oven dry.

3, experimental implementation

Extracting solution A method extracts the step of Chinese Fir Leaves and root total serum IgE:

(1) in 1.5 ml centrifuge tubes, following liquid is mixed: 1 ml extracting solution and 50 μ l mercaptoethanols, be designated as extracting solution A; (CTAB method needs extracting solution preheating 1h in 65 DEG C of water-baths);

(2) get the fresh Chinese Fir Leaves of 100mg and root is put in 2 ml centrifuge tubes respectively, and respectively add 25 mm steel balls, be put into rapidly quick-frozen in liquid nitrogen, use high-throughput tissue grinder grind into powder;

(3) in the centrifuge tube after grinding, add rapidly 1 ml extracting solution A, vortex mixes, and room temperature leaves standstill 10 min, 4 DEG C, centrifugal 15 min of 13000g; (CTAB method needs to place 20 min in 65 DEG C of water-baths, period needs put upside down mixing 1 time every 5 min.）

(4) get supernatant 600 μ l that step (3) obtains in new 1.5 ml centrifuge tubes, add 300 μ l 5 mol/L NaCl and 500 μ l chloroforms, mixing, 4 DEG C, centrifugal 5 min of 13000g;

(5) get supernatant 600 μ l that step (4) obtains in new 1.5 ml centrifuge tubes, add isopyknic water-saturated phenol: chloroform (1:1, v/v), mixing, 4 DEG C, centrifugal 5 min of 13000g;

(6) get supernatant 400 μ l that step (5) obtains in new 1.5 ml centrifuge tubes, add the Virahol of equal-volume precooling, on side-sway shaking table, shake up 10 min(room temperatures carry out); (CTAB method LiCl precipitates needs 4 DEG C and precipitates at least 6h, even spends the night).

(7) be drawn in centrifugal column by step (6) described liquid, 4 DEG C, centrifugal 1 min of 13000g, abandons liquid;

(8) ethanol adding 500 μ l 75% precoolings in the centrifugal column described in step (7) washes precipitation, 4 DEG C, centrifugal 1 min of 13000g;

(9) liquid is abandoned, repeating step (8);

(10) liquid is abandoned, 4 DEG C, empty centrifugal 1 min of 13000g;

(11) in exchonge step (10) collection tube of bottom be new 1.5 ml without RNA enzyme centrifuge tube, add 50 μ l DEPC water to above-mentioned centrifugal column central authorities, leave standstill 2 min, 4 DEG C, centrifugal 1 min of 13000g;

(12) liquid bottom centrifugal rear centrifuge tube is sucked in centrifugal column again, leave standstill 2 min, 4 DEG C, centrifugal 1 min of 13000g;

(13) abandon centrifugal column, and carry out mark-20 DEG C save backup.

4, RNA integrity detection

5 μ l RNA solution prepared by the method for getting the invention described above and 1 μ l 6 × loading buffer mix, and the agarose gel electrophoresis with 1% detects its integrity.Can observe than more complete RNA by gel imaging, wherein the brightness of 28S RNA is approximately the twice of 18S RNA, and well is normal, without shinny conditions of streaking (Fig. 1).The electrophoresis result of the Lignum seu Ramulus Cunninghamiae Lanceolatae leaf that CTAB method is extracted and root is shown in Fig. 2, can see obvious DNA pollution, without 5S RNA.

5, RNA quality examination

Get 2 μ l RNA solution prepared by aforesaid method, join in TECAE microplate reader and measure RNA concentration and OD _260/280ratio, analyze RNA quality.Result shows that the RNA that present method is extracted in the Chinese Fir Leaves higher containing phenols and root has higher Quality and yield, wherein OD _260/280value is respectively 2.06,2.04,2.06,1.97(from left to right), output be 67.3,61.26,58.58,63.62 μ g/g(from left to right).

Note: CTAB method extracts the step of Chinese Fir Leaves and root total serum IgE:

(1) 2%CTAB of 600ul is added in 1.5ml centrifuge tube, and adds the beta-mercaptoethanol of 50uL, vibration mixing, 65 DEG C of water-bath preheatings;

(2) get the fresh Chinese Fir Leaves of 100 mg and root is put in 2 ml centrifuge tubes respectively, and respectively add 25 mm steel balls, be put into rapidly quick-frozen in liquid nitrogen, use high-throughput tissue grinder grind into powder;

(3) material powder is shifted as in 2% CTAB centrifuge tube of 65 DEG C of water-bath preheatings, mixing, and in 65 DEG C of water-bath 20 min, period puts upside down once every 5 min tendernesses;

(4) phenol of 600 ul is added: chloroform: primary isoamyl alcohol (25:24:1), shaken well, 4 DEG C, centrifugal 10 min of 12000 g;

(5) shift supernatant in another 1.5 ml centrifuge tube, add isopyknic chloroform: put upside down mixing after primary isoamyl alcohol (24:1), 4 DEG C, centrifugal 10 min of 12000 g;

(6) supernatant is transferred in another 1.5 ml centrifuge tube, add 10 M LiCl of 1/4 volume, make its final concentration be 2 mol/L, after tenderness puts upside down mixing, 4 DEG C of precipitates overnight;

(7) 4 DEG C, centrifugal 20 min of 12000 g, abandon supernatant;

(8) add 500 μ l SSTE dissolution precipitations, add 500 μ l chloroform;

(9) 4 DEG C, centrifugal 10 min of 12000 g;

(10) get supernatant, add dehydrated alcohol and make its final concentration be 70%, place 1 h for-20 DEG C;

(11) 4 DEG C, centrifugal 20 min of 12000 g;

(12) abandon supernatant, add 75% washing with alcohol precipitation, 4 DEG C, centrifugal 10 min of 12000 g;

(13) abandon supernatant, siphon away unnecessary alcohol with pipettor, dry 5 ~ 10 min, add 50 μ l DEPC water dissolution precipitations.

The OD of the Lignum seu Ramulus Cunninghamiae Lanceolatae leaf that CTAB method is extracted and root _260/280value is respectively 2.10,2.12, and output is 45.3,39.9 μ g/g.

  

Claims (3)

1. a method for rapid extraction China fir total serum IgE, is characterized in that: comprise the following steps:

(1) in 1.5 ml centrifuge tubes, following liquid is mixed: 1 ml SDS extracting solution and 40-60 μ l mercaptoethanol, be designated as extracting solution A;

(2) get the fresh China fir material of 90-120mg to be put in 2 ml centrifuge tubes, and add steel ball, be put into rapidly quick-frozen in liquid nitrogen, use high-throughput tissue grinder grind into powder;

(3) in the centrifuge tube after grinding, add rapidly 0.8-1.5 ml extracting solution A, vortex mixes, and room temperature leaves standstill 8-15 min, 4 DEG C, the centrifugal 10-20 min of 13000g;

(4) draw in 1.5 new ml centrifuge tubes of 500-700 μ l supernatant to, add 200-400 μ l 5 mol/L NaCl and 400-600 μ l chloroform, mixing, 4 DEG C, the centrifugal 4-7 min of 13000g;

(5) draw 500-700 μ l supernatant, add isopyknic water-saturated phenol: the mixed solution of chloroform v/v=1:1, mixing, 4 DEG C, the centrifugal 4-7 min of 13000g;

(6) draw 300-500 μ l supernatant in new 1.5 ml centrifuge tubes, add the Virahol of equal-volume precooling, under room temperature, on side-sway shaking table, shake up 10 min;

(7) liquid after above-mentioned shaking up is drawn in centrifugal column, 4 DEG C, the centrifugal 1-3 min of 13000g;

(8) the washing with alcohol precipitation of 75% precooling is added, 4 DEG C, the centrifugal 1-3 min of 13000g;

(9) repeating step (8);

(10) liquid is abandoned, 4 DEG C, the empty centrifugal 1-3 min of 13000g;

(11) collection tube changing bottom be new 1.5 ml without RNA enzyme centrifuge tube, add 30-50 μ l DEPC water to above-mentioned centrifugal column central authorities, leave standstill 2-5 min, 4 DEG C, the centrifugal 1-3 min of 13000g;

(12) liquid bottom centrifugal rear centrifuge tube is sucked in centrifugal column again, leave standstill 2-5 min, 4 DEG C, the centrifugal 1-3 min of 13000g;

(13) abandon centrifugal column, new collection tube-20 DEG C is saved backup.

2. the method for described rapid extraction China fir total serum IgE according to claim 1, is characterized in that: the configuration of the described SDS extracting solution of step (1): 0.25M NaCl, 0.05M Tris-HCl, pH=7.5,20mM EDTA, pH=8.0,1%SDS.

3. the method for described rapid extraction China fir total serum IgE according to claim 1, is characterized in that: comprise the following steps:

(1) in 1.5 ml centrifuge tubes, following liquid is mixed: 1 mlSDS extracting solution and 50 μ l mercaptoethanols, be designated as extracting solution A;

(2) get the fresh China fir material of 100mg to be put in 2 ml centrifuge tubes, and add 25 mm steel balls, be put into rapidly quick-frozen in liquid nitrogen, use high-throughput tissue grinder grind into powder;

(3) in the centrifuge tube after grinding, add rapidly 1 ml extracting solution A, vortex mixes, and room temperature leaves standstill 10 min, 4 DEG C, centrifugal 15 min of 13000g;

(4) draw in 1.5 new ml centrifuge tubes of 600 μ l supernatants to, add 300 μ l 5 mol/L NaCl and 500 μ l chloroforms, mixing, 4 DEG C, centrifugal 5 min of 13000g;

(5) draw 600 μ l supernatants, add isopyknic water-saturated phenol: the mixed solution of chloroform v/v=1:1, mixing, 4 DEG C, centrifugal 5 min of 13000g;

(6) draw 400 μ l supernatants in new 1.5 ml centrifuge tubes, add the Virahol of equal-volume precooling, under room temperature, on side-sway shaking table, shake up 10 min;

(7) liquid after above-mentioned shaking up is drawn in centrifugal column, 4 DEG C, centrifugal 1 min of 13000g;

(8) the washing with alcohol precipitation of 500 μ l 75% precoolings is added, 4 DEG C, centrifugal 1 min of 13000g;

(9) repeating step (8);

(10) liquid is abandoned, 4 DEG C, empty centrifugal 1 min of 13000g;

(11) collection tube changing bottom be new 1.5 ml without RNA enzyme centrifuge tube, add 50 μ l DEPC water to above-mentioned centrifugal column central authorities, leave standstill 2 min, 4 DEG C, centrifugal 1 min of 13000g;

(12) liquid bottom centrifugal rear centrifuge tube is sucked in centrifugal column again, leave standstill 2 min, 4 DEG C, centrifugal 1 min of 13000g;

(13) abandon centrifugal column, new collection tube-20 DEG C is saved backup.