CN107603975A - A kind of lily method for extracting total RNA - Google Patents
A kind of lily method for extracting total RNA Download PDFInfo
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- CN107603975A CN107603975A CN201711116514.3A CN201711116514A CN107603975A CN 107603975 A CN107603975 A CN 107603975A CN 201711116514 A CN201711116514 A CN 201711116514A CN 107603975 A CN107603975 A CN 107603975A
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Abstract
The invention discloses a kind of lily method for extracting total RNA, is on the basis of traditional Trizol methods, using chloroform to sample cleanup impurity elimination 3 times, and RNA precipitate is washed 2 times using 75% ethanol.The inventive method can not only preferably remove the impurity in RNA sample, and the RNA concentration extracted is high, available for each kind of extraction lily, the high quality RNA of each organ.
Description
Technical field
The invention belongs to technical field of molecular biology, is related to a kind of RNA extracting method, optimizes more particularly to one kind
Plant lily method for extracting total RNA.
Background technology
Total serum IgE is extracted from plant, is the important prerequisite for carrying out plant research work.The RNA of high quality is follow-up RT-
PCR and gene cloning, the basis of expression.The method for extracting total RNA of comparative maturity has Trizol methods, CTAB methods and reagent at present
Box method.
Trizol methods and RNA isolation kit extraction RNA test operations are simple, are various species extraction RNA prefered methods.But
For the species material rich in polysaccharide and polyphenol, pollution be present with Trizol methods extraction RNA, and the RNA of RNA isolation kit extraction is then
Concentration is too low.Equally, the deficiency taken using CTAB methods there is also complex operation, easily polluted, it is impossible to be used in subsequent gene table
Up to analysis and gene cloning etc..
Lily is rich in polysaccharide and polyphenol substance in respectively organizing.Because many physicochemical properties and RNA of polysaccharide are much like, so as to
When precipitating RNA, the gelatinous precipitate rich in polysaccharide is often produced.This RNA precipitate containing polysaccharide is insoluble in water or molten
The solution of sticky shape is produced after solution；And if using impurity elimination is repeated, while Polysaccharide removing, RNA also easy quilts
Take away, cause the reduction of RNA yield.Therefore, a large amount of polysaccharide present in lily have impact on extraction RNA quality and concentration.
Such a problem all be present in fact, being extracted rich in the RNA of polysaccharide and polyphenol substance tissue, or extracting method is time-consuming
Take a lot of work, RNA easily pollutes, or the RNA amounts obtained are very little, it is impossible to meets the needs of follow-up study.Therefore, it is necessary to develop one kind
For the method for extracting total RNA containing polysaccharide polyphenol material, with meet from it is including lily, rich in polysaccharide polyphenol organ
Extract the demand of high quality RNA.
The content of the invention
It is an object of the invention to provide a kind of lily method for extracting total RNA, this method is suitable including through including lily, rich
The Total RNAs extraction of the organ containing polysaccharide polyphenol.
Lily method for extracting total RNA of the present invention includes：
1）Take lily sample to be placed in liquid nitrogen, be ground into fine powder, the ratio for adding 0.1g samples with every ml lysates adds lysate
In, 3min is shaken, centrifugation, takes supernatant to stand, is kept completely separate nucleic acid-protein compound；
2）The chloroform of the supernatant volume 20% is added in supernatant, after being stored at room temperature 3min, acutely concussion centrifugation, takes supernatant,
Chloroform is added, is repeated 3 times；
3）Supernatant is taken, adds the isopropanol of supernatant volume 50%, after being stored at room temperature 10min, is centrifuged after reverse mixing, reject supernatant,
Retain bottom precipitation；
4）75% isometric ethanol is added in precipitation, overturns and mixes, fully washing, centrifugation, adds 75% ethanol washing 1
It is secondary, spontaneously dry；
5）By dry precipitation with the DEPC-ddH no less than 30 μ L2O dissolves, and obtains RNA extract solutions.
Wherein, described lily sample be by lily sample it is in vitro after be placed on ice, -80 DEG C of refrigerators are placed in after liquid nitrogen flash freezer
The sample of preservation.
In said extracted method of the present invention, the lysate can be GenStar TRIGene, TaKaRa RNAiso
Lysate used in the various conventional Trizol methods such as Plus.
In the operational manual of Trizol extraction methods, purify the operation of sample using chloroform and RNA precipitate is washed
Wash process only to carry out once, do not repeat.Therefore, completing whole RNA extraction process only needs 3h.But fully according to
Found after the RNA of Trizol specifications method extraction lily, not only purity is inadequate by the RNA that this method is obtained, and pollutes tight
Weight.
And RNA isolation kit extraction RNA process merges the cracking of sample together with purification impurity elimination step, thus lead
Cause the quantity of single treatment sample to reduce, although the used time is shorter, only about 2h, can only obtain 20ng's or so per 1.5mL pipes
RNA, such amount are insufficient for the needs of follow-up study.
The present invention extracts lily total serum IgE by the Trizol methods of improvement, and in the extracting method, the 2nd step is using chloroform to sample
Product purification impurity elimination 3 times, the 4th step is washed 2 times to RNA precipitate, not only preferably eliminates the impurity in RNA sample, and often
1.5mL pipes can obtain 100ng or so RNA.
Extracting method of the present invention is simple to operate, efficiency high, simple using reagent, and extraction RNA mass is high, concentration is high, can use
In each kind of extraction lily, the high quality RNA of each organ.
Brief description of the drawings
Fig. 1 is the electrophoretogram that embodiment 1 extracts lily total serum IgE.
Fig. 2 is the electrophoretogram that embodiment 2 extracts lily total serum IgE.
Fig. 3 is the electrophoretogram that comparative example 1 extracts lily total serum IgE.
Fig. 4 is the electrophoretogram that comparative example 2 extracts lily total serum IgE.
Embodiment
The present invention is described in further detail below by way of specific embodiment.
In following each embodiment extraction process, used various utensils, including centrifuge tube, pipette tips, mortar, pestle, key
Spoon etc., it is intended to the 20min that first sterilized at 121 DEG C.
75% ethanol needed in extraction process, uses DEPC-ddH2O is configured.
The lily sample that each embodiment needs be by the sample of collection it is in vitro after be placed on ice, -80 are placed in after liquid nitrogen flash freezer
The sample preserved in DEG C refrigerator.
Embodiment 1
The present embodiment uses the RNA in GenStar TRIGene total RNA extraction reagents box extraction lily.
Concrete operations flow is：
1st, 1mL GenStar TRIGene total RNA extraction reagents are added in 1.5mL centrifuge tubes, are placed on ice.
2nd, lily sample is ground in liquid nitrogen, sample will be constantly in freezing state in process of lapping, can not thaw.Take
Sample ground 0.1g or so is added in above-mentioned centrifuge tube, and 3min is shaken on turbula shaker.
3rd, 12000 × g centrifugations 10min at 4 DEG C, draws supernatant into new centrifuge tube.
4th, pyrolysis product places 5min in room temperature, is kept completely separate nucleic acid-protein compound.
5th, 0.2mL chloroforms are added, acutely shake 15s, room temperature places 3min.
6th, 12000 × g centrifugations 15min, sample are divided into three layers at 4 DEG C：Red lower floor, intermediate thin film layer, and color with
Sample and the upper strata become.
7th, upper strata is drawn into a new centrifuge tube, about 0.5mL.
8th, step 5~7 are repeated twice, wherein the upper strata volume integral drawn out every time Wei not 0.4mL and 0.3mL.
9th, 0.5mL isopropanols are added, overturns and mixes, room temperature places 10min.
10th, 12000 × g centrifugations 10min at 4 DEG C, reject supernatant, obtains gluey RNA precipitate.
11st, the ethanol of 1mL 75% is added, overturns and mixes, washing precipitation.
12nd, 7500 × g centrifugations 5min, reject supernatant at 4 DEG C.
13rd, repeat step 11~12 is once.
14th, residual liquid in centrifuge tube is drawn with liquid-transfering gun, room temperature dries 10~20min.
15th, 30 μ L DEPC-ddH are added2RNA is dissolved in O, liquid-transfering gun piping and druming for several times.
16th, detected by RNA electrophoresis and nucleic acid instrument.
17th, -80 DEG C of preservations, avoid multigelation after dispensing.
Above operating process takes 4.5h altogether.
Fig. 1 gives the electrophoretogram of the lily total serum IgE of the present embodiment extraction, it can be seen that RNA is both without degraded
(no conditions of streaking), also pollution-free (loading wells noresidue).And then using nucleic acid instrument detect the present embodiment obtain RNA concentration as
93.1ng/µL.Illustrate that the present embodiment method is applied to the extraction of lily total serum IgE.
Embodiment 2
The present embodiment uses the TRIGene lysates in TaKaRa RNAiso Plus lysates alternate embodiment 1, specific behaviour
Make that flow is identical with embodiment 1, the upper strata volume integral drawn out every time in step 8 Wei not 0.35mL and 0.25mL.Operation
Flow takes 4.5h altogether.
Extraction effect is as shown in Fig. 2 nucleic acid instrument detection RNA concentration is 143.6ng/ μ L.The RNA of the present embodiment extraction is same
Both it is (no conditions of streaking) or pollution-free (loading wells noresidue) without degrading, suitable for the extraction of lily total serum IgE.
Comparative example 1（RNA isolation kit）
The present embodiment was specifically extracted using Tiangeng plant total RNA extraction reagent box (RNAprep Pure) extraction lily total serum IgE
Journey follows the steps below.
1st, 50~100mg lily plant leaf blades are taken, the rapid grind into powder in liquid nitrogen, 450 μ l RL of addition (addition β-
Mercaptoethanol), the acutely concussion that is vortexed mixes.
2nd, all solution are transferred on Filter column CS (Filter column CS is placed in collecting pipe), with 12000rpm centrifugation 2~
5min, careful supernatant of drawing in collecting pipe is into RNase-Free centrifuge tube.
3rd, the absolute ethyl alcohol of 0.5 times of supernatant volume is slowly added to, is mixed, obtained solution is transferred to absorption together with precipitation
In post CR3,12000rpm centrifuges 30~60s, outwells waste liquid in collecting pipe, adsorption column CR3 is put back in collecting pipe.
4th, 350 μ l protein liquid removals RW1,12000rpm 30~60s of centrifugation are added into adsorption column CR3, are outwelled in collecting pipe
Waste liquid, adsorption column CR3 is put back in collecting pipe.
5th, 80 μ l DNase I working solutions are added to adsorption column CR3 centers, room temperature places 15min.
6th, 350 μ l protein liquid removals RW1,12000rpm 30~60s of centrifugation are added into adsorption column CR3, are outwelled in collecting pipe
Waste liquid, adsorption column CR3 is put back in collecting pipe.
7th, 500 μ l rinsing liquid RW are added into adsorption column CR3, are stored at room temperature 2min, 12000rpm centrifuges 30~60s, falls
Fall waste liquid in collecting pipe, adsorption column CR3 is put back in collecting pipe.
8th, repeat step 7.
9th, 12000rpm centrifuges 2min, outwells waste liquid.Adsorption column CR3 is placed in room temperature and places number min, thoroughly to dry suction
Remaining rinsing liquid in enclosure material.
10th, adsorption column CR3 is put into a new RNase-Free centrifuge tube, vacantly dripped to the middle part of adsorbed film
Add 30~100 μ l RNase-Free ddH2O, room temperature place 2min, 12000rpm centrifugation 2min, obtain RNA solution.
Above extraction process duration 2h.Fig. 3 provides the electrophoretic effects figure of extracted lily total serum IgE.With mark
(Marker) compare, RNA band brightness is significant lower, shows that RNA concentration is relatively low.The concentration that nucleic acid instrument detects to obtain RNA is only
33.8ng/ μ L or so.
Comparative example 2（General T rizol methods）
This comparative example uses GenStar TRIGene to extract lily total serum IgE as lysate with Trizol methods, specifically extracted
Journey follows the steps below.
1st, 1mL GenStar TRIGene total RNA extraction reagents are added in 1.5mL centrifuge tubes, are placed on ice.
2nd, lily sample is ground in liquid nitrogen, sample will be constantly in freezing state in process of lapping, can not thaw.Take
Sample ground 0.1g or so is added in above-mentioned centrifuge tube, and 3min is shaken on turbula shaker.
3rd, 12000 × g centrifugations 10min at 4 DEG C, draws supernatant into new centrifuge tube.
4th, pyrolysis product places 5min in room temperature, is kept completely separate nucleic acid-protein compound.
5th, 0.2mL chloroforms are added, acutely shake 15s, room temperature places 3min.
6th, 12000 × g centrifugations 15min, sample are divided into three layers at 4 DEG C：Red lower floor, intermediate thin film layer, and color with
Sample and the upper strata become.
7th, upper strata is drawn into a new centrifuge tube, about 0.5mL.
8th, 0.5mL isopropanols are added, overturns and mixes, room temperature places 10min.
9th, 12000 × g centrifugations 10min at 4 DEG C, reject supernatant, obtains gluey RNA precipitate.
10th, the ethanol of 1mL 75% is added, overturns and mixes, washing precipitation.
11st, 7500 × g centrifugations 5min, reject supernatant at 4 DEG C.
12nd, residual liquid in centrifuge tube is drawn with liquid-transfering gun, room temperature dries 10~20min.
13rd, 30 μ L DEPC-ddH are added2RNA is dissolved in O, liquid-transfering gun piping and druming for several times.
14th, detected by RNA electrophoresis and nucleic acid instrument.
15th, -80 DEG C of preservations, avoid multigelation after dispensing.
Above operating process takes 3h altogether.Extraction effect electrophoretogram illustrates RNA as shown in figure 4, loading wells residue is more
It is seriously polluted.
Above-mentioned 3 kinds of extracting methods relatively understand that general T rizol methods are simple to operate, but the RNA extracted has pollution.Examination
Although agent box method does not pollute, the amount of extraction is insufficient for the needs of subsequent research.Although extracting method duration of the present invention omits
It is long, but obtained RNA mass is high, concentration is high, on the one hand overcomes lily and respectively organizes because of polysaccharide polyphenol and the difficulty of RNA extraction difficulties
Topic, on the other hand, fund is saved because required reagent is simple.
Above-described embodiment is only the preferred technical solution of the present invention, is not used to carry out any restrictions to the present invention.For
For those skilled in the art, the present invention can have various modifications and variations.Within the spirit and principles of the invention, made
Any modification, equivalent substitution and improvements etc., should be included in the scope of the protection.
Claims (3)
1. a kind of lily method for extracting total RNA, including：
1）Take lily sample to be placed in liquid nitrogen, be ground into fine powder, the ratio for adding 0.1g samples with every ml lysates adds lysate
In, 3min is shaken, centrifugation, takes supernatant to stand, is kept completely separate nucleic acid-protein compound；
2）The chloroform of the supernatant volume 20% is added in supernatant, after being stored at room temperature 3min, acutely concussion centrifugation, takes supernatant,
Chloroform is added, is repeated 3 times；
3）Supernatant is taken, adds the isopropanol of supernatant volume 50%, after being stored at room temperature 10min, is centrifuged after reverse mixing, reject supernatant,
Retain bottom precipitation；
4）75% isometric ethanol is added in precipitation, overturns and mixes, fully washing, centrifugation, adds 75% ethanol washing 1
It is secondary, spontaneously dry；
5）By dry precipitation with the DEPC-ddH no less than 30 μ L2O dissolves, and obtains RNA extract solutions.
2. lily method for extracting total RNA according to claim 1, it is characterized in that described lily sample is by lily sample
It is placed on ice after in vitro, the sample that -80 DEG C of refrigerators preserve is placed in after liquid nitrogen flash freezer.
3. lily method for extracting total RNA according to claim 1, it is characterized in that the lysate be TRIGene or
RNAiso Plus。
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109666670A (en) * | 2019-01-18 | 2019-04-23 | 青岛农业大学 | A kind of Tsingtau lily floral organ total tissue RNA extracting method |
| CN111073887A (en) * | 2020-01-23 | 2020-04-28 | 西南林业大学 | Extraction method and application of paphiopedilum high-quality total RNA |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109666670A (en) * | 2019-01-18 | 2019-04-23 | 青岛农业大学 | A kind of Tsingtau lily floral organ total tissue RNA extracting method |
| CN111073887A (en) * | 2020-01-23 | 2020-04-28 | 西南林业大学 | Extraction method and application of paphiopedilum high-quality total RNA |
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