CN110106167A - A kind of lily bulb method for extracting total RNA suitable for Iso-Seq - Google Patents

Abstract

The present invention relates to molecular biology fields, it is desirable to provide a kind of lily bulb method for extracting total RNA suitable for Iso-Seq, comprising: by the grinding of blocky tissue culture bulb in powder, is added in lysate CLB and beta -mercaptoethanol mixed liquor, cracking to homogenate state；By increasing sample size and lysate volume to improve RNA concentration from total amount, and optimize cracking process by the way that beta -mercaptoethanol combination lysate and sequence of operations is added, effectively remove polysaccharide and phenols, dissolve out RNA preferably from tissue, column, which is removed, with the genome of three times again removes DNA respectively, RNA is concentrated finally by an adsorption column, further increases RNA concentration.The present invention more preferably protects the safety of operator without the use of toxic irritation reagents such as phenol, chloroforms；It is easy to operate, it is time-consuming short；The RNA integrality of extraction is good, purity is high, can be satisfied with the needs of most molecular biology experiments such as overall length transcript profile sequencing.

Description

A kind of lily bulb method for extracting total RNA suitable for Iso-Seq

Technical field

The invention belongs to molecular biology fields, and in particular to a kind of extraction method of lily bulb total serum IgE, it is especially suitable In the extraction of the lily bulb total serum IgE of Iso-Seq.

Background technique

The research of overall length transcript profile is an important channel for understanding living organism function, and Iso-Seq is more and more at present Applied to no reference genome species.Iso-Seq full name is called Isoform-sequencing, is that Pacbio company develops it Transcript sequencing technologies standardization name；It is that the long feature for reading length is sequenced using three generations, does not interrupt transcript, directly survey Sequence, to obtain a kind of sequencing technologies of overall length transcript.The technology can read the long mRNA full length sequence and complete of obtaining by overlength Whole structural information overcomes its transcript and splices short, the incomplete problem of information, is follow-up function gene excavating, gene cloning Equal researchs provide material base.However, Iso-Seq is to sample to be tested relative to molecular engineerings such as two generation sequencing technologies, PCR More stringent requirements are proposed for quality, concentration and the integrality of RNA.

Lily is famous flowering bulb, ornamental, edible and medical value with higher.Its genome is huge, at present Genome sequencing is not yet carried out, available gene sequence information is lacked, the utilization of Iso-Seq technology can be greatly promoted hundred Close research.Lily respectively organize in rich in the substances such as polysaccharide polyphenol, it is more prominent in bulb, and many physicochemical properties of polysaccharide and RNA is much like, to often generate the gelatinous precipitate for being rich in polysaccharide, this RNA precipitate containing polysaccharide when precipitating RNA Thick solution is generated after being insoluble in water, or dissolution, RNA is also easy to be pulled away while Polysaccharide removing, causes RNA yield Reduction, therefore from lily extract high quality RNA have comparable difficulty.

Lily method for extracting total RNA is the report about blade, petal mostly at present, and is suitable for lily bulb total serum IgE Extracting method it is seldom, only Du Yunpeng, Li, Xueyan etc. is few in number, and with modified CTAB method to extract lily bulb total RNA, but there are still part lilies to be difficult to the problems such as extracting and take a long time, is cumbersome, is not suitable for high-volume fast operating. TrizoL method extracts RNA degradation seriously and there is pollution；General reagent box method is easy to operate, the used time is short, but most extracting concentration It is too low.Therefore, there is an urgent need to develop it is a kind of it is convenient and easy, stability is good, suitable for the lily bulb Total RNAs extraction of Iso-Seq Method.

Summary of the invention

The technical problem to be solved by the present invention is to overcome deficiency in the prior art, provide a kind of being applicable in for fast and stable In the lily bulb method for extracting total RNA of Iso-Seq.The lily bulb total serum IgE integrality that this method is extracted is good, purity is high, energy Enough solve the problems, such as that lily bulb Total RNAs extraction is difficult.

In order to solve the technical problem, solution of the invention is:

A kind of lily bulb method for extracting total RNA suitable for Iso-Seq is provided, comprising the following steps:

(1) article used in experimentation and equipment are carried out disinfection and is sterilized；

(2) sample for taking blocky tissue culture bulb is ground under conditions of maintaining temperature with liquid nitrogen；The powdered sample of gained Product pour into centrifuge tube, are placed in spare in liquid nitrogen；

(3) lysate CLB and beta -mercaptoethanol are sequentially added into new centrifuge tube；It is added after mixing spare powdered Sample, cracking to homogenate state；

(4) it to lysate centrifugal treating, takes supernatant that new centrifuge tube is added, the dehydrated alcohol of half volume is added；Piping and druming After mixing, genome is fed the mixture into immediately and removes column；To column centrifugation is removed, waste liquid is abandoned；

(5) genome removing column is put into new centrifuge tube, adds centrifugal treating after lysate RLT PLus；Collect filter Liquid, and volume is write down, the dehydrated alcohol of half volume is added, piping and druming mixes immediately；

(6) it feeds the mixture into adsorption column RA, adsorption column RA is centrifuged, abandon waste liquid；

(7) protein liquid removal RW1 is added into adsorption column RA, is centrifuged after being placed at room temperature for, abandons waste liquid；With rinsing liquid RW to absorption After column RA rinsing twice, then the centrifugation that carries out that treated；

(8) adsorption column RA is put into RNase free centrifuge tube, RNase free water elution is added, obtained total RNA solution.

In the present invention, disinfection and sterilizing in the step (1) include: that will test the pipette tips that need to be used, centrifuge tube, grind Alms bowl, pestle and spoon aluminium-foil paper package are placed in high-pressure sterilizing pot, 121 DEG C of sterilizing 40min；80 DEG C are subsequently placed in baking oven 30min is dried, it is spare；After operating table surface is sprayed with 75% alcohol, wiped with aseptic paper dry；Operator wears disposable sterilized mask And gloves, and replace in due course.

It in the present invention, maintains temperature to refer to liquid nitrogen in the step (2), grinds mortar used and be put into liquid nitrogen in advance Pre-cooling, repeatedly appropriate addition liquid nitrogen is to keep low temperature during the grinding process, and preventing sample from deliquescing, (otherwise RNA is easy degradation and sample Product are easy frosting, and the later period is easy conglomeration or agglomeration in lysate and influences lytic effect).

In the present invention, in the step (3), lysate CLB, beta -mercaptoethanol and sample are added in following ratios: cracking Liquid CLB volume 1.6mL: beta -mercaptoethanol volume 80 μ L: 400~500mg of sample quality；Wherein, the volume ratio of beta -mercaptoethanol Concentration is 5%.

In the present invention, in the step (3), the cracking refers to: sample is added and is acutely vortexed immediately, vortex time is 1~2min is blown and beaten if there is the cotton-shaped agglomerate of indissoluble with pipette tips to assist its cracking；65 DEG C of water-baths are put back in cracking process 15min takes out 2~3 Assisted Cleavages that are vortexed during water-bath；Until without obvious agglomerating or blocking floccule.

(there are many schemes for cracking operation, such as can choose and be all added to a 2mL centrifuge tube mixing mixing, can also It is each that 200~250mg of sample, lysate CLB 0.8mL, 40 μ L of beta -mercaptoethanol is added to select two 1.5mL centrifuge tubes Supernatant is transferred in the same new centrifuge tube after mixing centrifugation with liquid-transfering gun by (volume by volume concentration 5%).)

In the present invention, in the step (4), mixture is averagely added to multiple genomes and removes column, and is centrifuged respectively；Add When entering mixture, is chosen with liquid-transfering gun and remove cotton-shaped insoluble floating material in mixture (to reduce the removing pressure that genome removes column Power more effectively removes DNA).

In the present invention, in the step (5), the additive amount of lysate RLT Plus is 500 μ L, and centrifugal speed is 13000rpm, centrifugation time are 30 seconds.

In the present invention, in the step (6), entire mixture is added in the same adsorption column RA, is guaranteed by concentration The concentration of RNA；When to adsorption column RA centrifugation, revolving speed 13000rpm, time 2min, and ensure liquid all filters after centrifugation Out, it is not remained on film.

In the present invention, in the step (7), specifically includes the following steps:

(7.1) add 700 μ L protein liquid removal RW1 into adsorption column RA, be placed at room temperature for 1min, 13000rpm is centrifuged 30 seconds, is abandoned Fall waste liquid；

(7.2) 500 μ L rinsing liquid RW, 13000rpm is added centrifugation 30 seconds, discards waste liquid；Add 500 μ L rinsing liquid RW Repeat the operation；It is added into dehydrated alcohol in advance (i.e. directly on the basis of original reagent 10mL by 10:42 volume ratio in rinsing liquid RW Add 42mL dehydrated alcohol)；

(7.3) adsorption column RA being put back in sky collecting pipe, 13000rpm is centrifuged 2min, as far as possible removing rinsing liquid, in order to avoid drift Washing lotion residual ethanol inhibits downstream reaction.

In the present invention, in the step (8), specifically includes the following steps:

(8.1) RNase free water is placed in 75 DEG C of water-baths in advance and preheats；

(8.2) adsorption column RA is put into RNase free centrifuge tube, 30 μ LRNase are added in position among adsorbed film Free water, affords total rna solution；

(8.3) total rna solution is placed in be measured on ice, or is placed in -80 DEG C of refrigerator and is saved.

Compared with prior art, the beneficial effects of the present invention are:

1, the present invention more preferably protects the safety of operator without the use of toxic irritation reagents such as phenol, chloroforms；

2, operation of the present invention is simple, time-consuming short；

3, the RNA integrality that the present invention extracts is good, purity is high, can be satisfied with most molecules such as overall length transcript profile sequencing The needs of biological experiment.

Detailed description of the invention

Fig. 1 is that embodiment 3 repeats hundred tissue culture bulb Nanodrop of open country detection RNA mass peak figure in 1；

Fig. 2 is the agarose gel electrophoresis figure of three kinds of lily bulb RNA in embodiment 1,2,3, from left to right successively are as follows: rope Nation, huge hundred and open country hundred, each kind (or kind) has 2 repetitions；

Specific embodiment

Below with reference to examples and drawings, the present invention is described in further detail, but embodiments of the present invention are unlimited In this.

The present invention is by three embodiments and two comparative examples to oriental hybrid lily ' Suo Bang ' (LiLium OrientaL Hybrid'Sorbonne'), huge ball lily (LiLium brownii var.giganteum) and rattlebush (LiLium Brownii) the Total RNAs extraction process of tissue culture bulb is illustrated (hereinafter with referred to as " Suo Bang ", " huge hundred " and " open country hundred " generation It replaces, wherein open country hundred is even more that lily bulb RNA is difficult to the Typical Representative extracted, in addition to this various method extractions fail to reach to want It asks).Each embodiment is respectively provided with 2 repetitions, and comparative example is not provided with repeating.

Preparation includes: to wrap up used pipette tips, centrifuge tube, mortar, pestle and spoon with aluminium-foil paper before testing 121 DEG C of sterilizing 40min in high-pressure sterilizing pot afterwards take out later and are put into 80 DEG C of baking 30min in baking oven, for use；Operating table surface is used It is wiped with aseptic paper dry after 75% alcohol is spraying, and gets out sterile mask and gloves.

Embodiment 1:

Rope nation tissue culture bulb total serum IgE is extracted with this method, comprising the following steps:

(1) Qu Suo nation tissue culture bulb fritter (- 80 DEG C of refrigerators are taken out) is put into the mortar that liquid nitrogen is pre-chilled in advance, is quickly ground Mill, during which adding liquid nitrogen in time prevents sample from deliquescing, and is ground to after uniform pulverulence and pours into 50mL centrifuge tube rapidly, will be from Heart pipe is put into spare in liquid nitrogen；

(2) take in 1.6mL lysate CLB to 2mL centrifuge tube (2 repetitions, i.e., 2 pipe, operate below for the sake of clarity be illustrated with Single repetitive operation is illustrated), 80 μ L (volume by volume concentration 5%) of beta -mercaptoethanol is added in lysate CLB, overturns mixed Even, the rope nation bulb powder for shifting about 400mg with the spoon of Liquid nitrogen precooler is acutely vortexed and auxiliary with pipette tips immediately into lysate Help piping and druming 1min, put back to 15min in 65 DEG C of water-baths in short-term, centre take out obtain for vortex 2 times being satisfied with homogenization effect (without not agglomerating or Blocking floccule)；

(3) lysate 13000rpm is centrifuged 10min；

(4) it takes lysate supernatant to go to a new centrifuge tube, the dehydrated alcohol of supernatant volume half is added, blows and beats immediately It mixes, not be centrifuged；

(5) 3 genomes are averagely added in mixture (being no more than 720 μ L every time) immediately to remove in column, remove column and is put into In collecting pipe, 13000rpm is centrifuged 2min, discards waste liquid, carefully chooses out flocculent undissolved substance with pipette tips when mixture is added；

(6) genomic DNA removing pillar is placed in a new 2mL centrifuge tube, is removed in genome and adds 500 μ L in column Lysate RLT PLus, 13000rpm is centrifuged 30 seconds, collects filtrate, and write down volume, the dehydrated alcohol of half volume is added, vertical I.e. piping and druming mixes；

(7) it feeds the mixture at once in an adsorption column RA (adsorption column is put into collecting pipe), 13000rpm centrifugation 2min discards waste liquid, it is ensured that liquid all filters out；

(8) add 700 μ L protein liquid removal RW1, be placed at room temperature for 1min, 13000rpm is centrifuged 30 seconds, discards waste liquid；

(9) 500 μ L rinsing liquid RW (dehydrated alcohol has been added) are added, 13000rpm is centrifuged 30 seconds, discards waste liquid, is added 500 μ L rinsing liquid RW come again；

(10) adsorption column RA is put back in sky collecting pipe, 13000rpm is centrifuged 2min；

(11) it takes out adsorption column RA to be put into a RNase free centrifuge tube, position adds 30 μ among adsorbed film LRNase free water (is heated in 75 DEG C of water-baths) in advance, is stored at room temperature 1min, and 12000rpm is centrifuged 1min, obtains RNA Solution carries out the items of Nanodrop detection, agarose gel electrophoresis and Hua Da company for Iso-Seq sequencing to it later Detection, concrete outcome are shown in attached drawing 2 and table 1.

Embodiment 2:

Huge hundred tissue cultures bulb total serum IgE is extracted with this method, comprising the following steps:

(1) it takes huge hundred tissue cultures bulb fritter (- 80 DEG C of refrigerators are taken out) to be put into the mortar that liquid nitrogen is pre-chilled in advance, quickly grinds Mill, during which adding liquid nitrogen in time prevents sample from deliquescing, and is ground to after uniform pulverulence and pours into 50mL centrifuge tube rapidly, will be from Heart pipe is put into spare in liquid nitrogen；

(2) take in 1.6mL lysate CLB to 2mL centrifuge tube (2 repetitions, i.e., two pipe, operate be illustrated for the sake of clarity below It is illustrated with single repetitive operation), 80 μ L (volume by volume concentration 5%) of beta -mercaptoethanol is added in lysate CLB, overturns mixed Even, the huge ball lily bulb powder for shifting about 450mg with the spoon of Liquid nitrogen precooler is acutely vortexed immediately into lysate 1.5min, puts back to 15min in 65 DEG C of water-baths in short-term, and centre takes out vortex 3 times and obtains being satisfied with homogenization effect (without agglomerating or blocking Floccule)；

(3) lysate 13000rpm is centrifuged 10min；

(4) it takes lysate supernatant to a new centrifuge tube, the dehydrated alcohol of supernatant volume half is added, piping and druming is mixed immediately It is even；

Step (5) step (5) into (10) and embodiment one arrives (10) unanimously, and details are not described herein.

(11) it takes out adsorption column RA to be put into a RNase free centrifuge tube, position adds 30 μ among adsorbed film LRNase free water (is heated in 75 DEG C of water-baths) in advance, is stored at room temperature 1min, and 12000rpm is centrifuged 1min, obtains RNA Solution carries out the items of Nanodrop detection, agarose gel electrophoresis and Hua Da company for Iso-Seq sequencing to it later Detection, concrete outcome are shown in attached drawing 2 and table 1.

Embodiment 3:

Wild hundred tissue culture bulb total serum IgEs are extracted with this method, comprising the following steps:

(1) it takes wild hundred tissue culture bulb fritters (- 80 DEG C of refrigerators are taken out) to be put into the mortar that liquid nitrogen is pre-chilled in advance, quickly grinds Mill, during which adding liquid nitrogen in time prevents sample from deliquescing, and is ground to after uniform pulverulence and pours into 50mL centrifuge tube rapidly, will be from Heart pipe is put into spare in liquid nitrogen；

(2) take in 1.6mL lysate CLB to 2mL centrifuge tube (2 repetitions, i.e., two pipe, operate be illustrated for the sake of clarity below It is illustrated with single repetitive operation), 80 μ L (volume by volume concentration 5%) of beta -mercaptoethanol is added in lysate CLB, overturns mixed It is even, with the rattlebush bulb powder of the spoon of Liquid nitrogen precooler transfer about 500mg into lysate, violent vortex 2min immediately, side With the cotton-shaped agglomerate of pipette tips piping and druming indissoluble, 15min in 65 DEG C of water-baths is put back in short-term, and centre takes out vortex 3 times and obtains being satisfied with homogenate Effect (without agglomerating or blocking floccule)；

(3) lysate 13000rpm is centrifuged 10min, precipitates the fragment that cannot be cracked；

(4) it takes lysate supernatant to go to a new centrifuge tube, the dehydrated alcohol of supernatant volume half is added, blows and beats immediately It mixes, not be centrifuged；

Step (5) step (5) into (10) and embodiment one arrives (10) unanimously, and details are not described herein.

(11) it takes out adsorption column RA to be put into a RNase free centrifuge tube, position adds 30 μ among adsorbed film LRNase free water (is heated in 75 DEG C of water-baths) in advance, is stored at room temperature 1min, and 12000rpm is centrifuged 1min, obtains RNA Solution carries out the items of Nanodrop detection, agarose gel electrophoresis and Hua Da company for Iso-Seq sequencing to it later Detection, concrete outcome are shown in attached drawing 1,2 and table 1.

Comparative example 1:

This comparative example extracts wild hundred bulb total serum IgEs using modified CTAB method, comprising the following steps:

(1) it takes wild hundred tissue culture bulb fritters (- 80 DEG C of refrigerators are taken out) to be put into the mortar that liquid nitrogen is pre-chilled in advance, quickly grinds Mill, during which adding liquid nitrogen in time prevents sample from deliquescing, and is ground to after uniform pulverulence and pours into 50mL centrifuge tube rapidly, will be from Heart pipe is put into spare in liquid nitrogen；

(2) 2mL centrifuge tube is taken, every pipe adds 700 μ L CTAB solution in draught cupboard, add 14 μ L beta -mercaptoethanols (2%), Each two pipe extracting solution of sample；

(3) test tube added is put into preheating in 65 DEG C of water-baths (slightly open, to pop in order to avoid pipe is heated)；

(4) ground hundred powder 150mg of open country is added in every pipe, and 65 DEG C of water-baths are put into after mixing well on whirlpool instrument 15min (centre, which is taken out, carries out whirlpool 3 times)；

(5) 720 μ L chloroforms: isoamyl alcohol reagent are added in every pipe in draught cupboard, 15 DEG C after whirlpool mixing, 10000rpm centrifugation 10min；

(6) suct clear liquid in draught cupboard and manage to new 2mL, then plus 720 μ L chloroforms: isoamyl alcohol reagent, whirlpool mix after 15 DEG C, 10000rpm is centrifuged 10min；

(7) Aspirate supernatant is managed to new 2mL, and the unification of two pipes is managed；

(8) 1/5 volume 12M LiCL is added, mixing is played in suction, and -20 DEG C of refrigerators place after 15h 4 DEG C, 12000rpm centrifugation 20min；

(9) supernatant is outwelled, dry, 20 μ L RNase-free water of addition, vortex mixing are buckled on blotting paper；

5 μ L 10 × DnaseI b μ ffer B μ ffer+1 μ L DnaseI+1.25 μ L are added in (10) 20 μ L samples 37 DEG C afterwards of RNaseO μ t+22.75 μ L RNase-free water (by sample number, calculating total amount, rear every 30 μ L/ sample is added) Metal bath 30min；

(11) 300 μ L RNase-free water are added, 350 μ L phenol: chloroform: isoamyl alcohol reagent are added, whirlpool mixes 4 DEG C afterwards, 12000rpm is centrifuged 5min；

(12) supernatant is taken, isometric chloroform is added: in the 1.5mL centrifuge tube of isoamyl alcohol reagent, 4 DEG C after whirlpool mixing, 10000rpm is centrifuged 1min；

(13) supernatant is sucked out into new 1.5mL centrifuge tube, the dehydrated alcohol and 1/10 volume of two volumes is added 3M sodium acetate；It turns upside down mixing, 4 DEG C after -80 DEG C of placement 1h, 12000rpm is centrifuged 20min；

(14) supernatant is abandoned, with 70% ethanol washing, 4 DEG C after whirlpool mixing, 12000rpm is centrifuged 15min；

(15) supernatant is outwelled after repeating previous action, and back-off is got rid of in centrifuge fastly on blotting paper, and liquid relief is used Raffinate is sucked out in rifle, and (< 10min, to prevent overdrying) is dried in draught cupboard；

(16) be added 30 μ L RNase-free water obtain total rna solution, by obtained total rna solution in Nanodrop detects RNA concentration and OD 260/280 and OD 260/230, and testing result sees attached list 1.

Comparative example 2:

Wild hundred bulb total serum IgEs are extracted using the polysaccharide polyphenol plant total RNA extraction reagent box of Tiangeng, step is such as Under:

(1) it takes wild hundred tissue culture bulb fritters to be put into the mortar that liquid nitrogen is pre-chilled in advance, quickly grinds, during which timely annex solution Nitrogen prevents sample from deliquescing, and is ground to after uniform pulverulence and pours into 50mL centrifuge tube rapidly, is put into spare in liquid nitrogen；

(2) 2mL centrifuge tube is taken, every pipe adds 700 μ L SL lysates, adds 35 μ L mercaptoethanols (volume by volume concentration 5%), often A two pipe extracting solution of sample；

(3) ground open country hundred powder about 150mg is added in every pipe, and the acutely concussion that is vortexed immediately mixes；

(4) 12000rpm is centrifuged 2min；

(5) supernatant is transferred on Filter column CS (Filter column is placed on collecting pipe), 12000rpm is centrifuged 2min, careful to inhale Take the supernatant in collecting pipe into the centrifuge tube of new RNase-Free；

(6) it is slowly added to the dehydrated alcohol of 0.4 times of supernatant volume, is mixed, two obtained pipe solution and precipitating are turned together Enter in the same adsorption column CR3,12000rpm is centrifuged 15 seconds, outwells waste liquid, adsorption column is put back to collecting pipe；

Remaining steps are carried out according to Tiangeng polysaccharide polyphenol plant total RNA extraction reagent box specification, obtained total rna solution It is saved in -80 DEG C of refrigerators, send every detection to Hua Da gene for Iso-Seq sequencing later, testing result is shown in Table 1.

("/" shows that this does not measure to 1 present invention of table with other common methods extraction lily bulb RNA Contrast on effect； RIN is the abbreviation of RNA Integrity Number).

From table 1 it follows that being above 80ng/ μ using 6 sample lily bulb total rna concentrations that this method is extracted L, and most of concentration is more than 100ng/ μ L, and use the comparative example 1 of modified CTAB method and extracted using Tiangeng kit For concentration in comparative example 2 less than 40ng/ μ L, concentration is too low, it is difficult to meet the requirement of sequencing.OD 260/280 is equal in implementation column Between 2.0~2.2, OD 260/230 is above 1.6, shows the total serum IgE extracted with this method not by albumen and phenols The pollution of equal substances, polysaccharide removal is cleaner, and gained total serum IgE purity is higher.RIN value reacts the complete implementations of RNA, and numerical value is got over Show that sample integrity is higher close to 10, conversely, integrality is poorer, the RNA for meeting Iso-Seq generally requires RIN >=6.5.With The RNA integrality that this method is extracted is high, and in addition to the huge hundred RIN values for repeating 1 gained total serum IgE are 6.4 in embodiment 2, remaining is big The RIN value that open country hundred repeats 2 in 7.0, embodiment 3 is even as high as 8.0.Relative to other common methods, the present invention extract hundred It closes bulb total rna concentration and purity is higher, integrality is good, meet the needs of most molecular biology researches such as Iso-Seq.

Claims (10)

1. a kind of lily bulb method for extracting total RNA suitable for Iso-Seq, which comprises the following steps:

(1) article used in experimentation and equipment are carried out disinfection and is sterilized；

(2) sample for taking blocky tissue culture bulb is ground under conditions of maintaining temperature with liquid nitrogen；Gained powdered samples are fallen Enter centrifuge tube, is placed in spare in liquid nitrogen；

(3) lysate CLB and beta -mercaptoethanol are sequentially added into new centrifuge tube；Spare powdered sample is added after mixing Product, cracking to homogenate state；

(4) it to lysate centrifugal treating, takes supernatant that new centrifuge tube is added, the dehydrated alcohol of half volume is added；Piping and druming mixes Afterwards, genome is fed the mixture into immediately removes column；To column centrifugation is removed, waste liquid is abandoned；

(5) genome removing column is put into new centrifuge tube, adds centrifugal treating after lysate RLT PLus；Filtrate is collected, and Volume is write down, the dehydrated alcohol of half volume is added, piping and druming mixes immediately；

(6) it feeds the mixture into adsorption column RA, adsorption column RA is centrifuged, abandon waste liquid；

(7) protein liquid removal RW1 is added into adsorption column RA, is centrifuged after being placed at room temperature for, abandons waste liquid；With rinsing liquid RW to adsorption column RA After rinsing twice, then the centrifugation that carries out that treated；

(8) adsorption column RA is put into RNase free centrifuge tube, RNase free water elution is added, it is molten to obtain total serum IgE Liquid.

2. the method according to claim 1, wherein the disinfection and sterilizing in the step (1) include: that will test Pipette tips, centrifuge tube, mortar, pestle and the spoon aluminium-foil paper package that need to be used are placed in high-pressure sterilizing pot, 121 DEG C of sterilizings 40min；80 DEG C of baking 30min are subsequently placed in baking oven, it is spare；After operating table surface is sprayed with 75% alcohol, wiped with aseptic paper dry；Behaviour Make personnel and wear disposable sterilized mask and gloves, and replaces in due course.

3. being ground the method according to claim 1, wherein maintaining temperature to refer to liquid nitrogen in the step (2) Mortar used in grinding is put into liquid nitrogen in advance to be pre-chilled, and repeatedly appropriate addition liquid nitrogen prevents sample to keep low temperature during the grinding process It deliquesces.

4. the method according to claim 1, wherein in the step (3), lysate CLB, beta -mercaptoethanol and Sample is added in following ratios: lysate CLB volume 1.6mL: beta -mercaptoethanol volume 80 μ L: 400~500mg of sample quality； Wherein, the volume by volume concentration of beta -mercaptoethanol is 5%.

5. the method according to claim 1, wherein the cracking refers in the step (3): it is vertical that sample is added It is acutely vortexed, vortex time is 1 to 2min, is blown and beaten if there is the cotton-shaped agglomerate of indissoluble with pipette tips to assist its cracking； 65 DEG C of water-bath 15min are put back in cracking process, take out 2-3 Assisted Cleavage of vortex during water-bath；Until without obvious agglomerating Or blocking floccule.

6. the method according to claim 1, wherein mixture to be averagely added to multiple bases in the step (4) Because of a group removing column, and it is centrifuged respectively；When mixture is added, the cotton-shaped insoluble floating material removed in mixture is chosen with liquid-transfering gun.

7. the method according to claim 1, wherein in the step (5), the additive amount of lysate RLT Plus For 500 μ L, centrifugal speed 13000rpm, centrifugation time is 30 seconds.

8. the method according to claim 1, wherein entire mixture is added same in the step (6) In adsorption column RA, guarantee the concentration of RNA by concentration；When to adsorption column RA centrifugation, revolving speed 13000rpm, time 2min, And liquid all filters out after ensuring to be centrifuged, and does not remain on film.

9. the method according to claim 1, wherein in the step (7), specifically includes the following steps:

(7.1) add 700 μ L protein liquid removal RW1 into adsorption column RA, be placed at room temperature for 1min, 13000rpm is centrifuged 30 seconds, discards useless Liquid；

(7.2) 500 μ L rinsing liquid RW, 13000rpm is added centrifugation 30 seconds, discards waste liquid；Add 500 μ L rinsing liquid RW repetition The operation；It is added into dehydrated alcohol in advance by 10:42 volume ratio in rinsing liquid RW；

(7.3) adsorption column RA being put back in sky collecting pipe, 13000rpm is centrifuged 2min, as far as possible removing rinsing liquid, in order to avoid rinsing liquid Residual ethanol inhibits downstream reaction.

10. the method according to claim 1, wherein in the step (8), specifically includes the following steps:

(8.1) RNase free water is placed in 75 DEG C of water-baths in advance and preheats；

(8.2) adsorption column RA is put into RNase free centrifuge tube, 30 μ L RNase free are added in position among adsorbed film Water affords total rna solution；

(8.3) total rna solution is placed in be measured on ice, or is placed in -80 DEG C of refrigerator and is saved.