CN105838707A - Method for extraction of total RNA from pomacea canaliculata muscular tissue - Google Patents
Method for extraction of total RNA from pomacea canaliculata muscular tissue Download PDFInfo
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- CN105838707A CN105838707A CN201510021161.3A CN201510021161A CN105838707A CN 105838707 A CN105838707 A CN 105838707A CN 201510021161 A CN201510021161 A CN 201510021161A CN 105838707 A CN105838707 A CN 105838707A
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- pomacea canaliculata
- muscular tissue
- chloroform
- isoamyl alcohol
- centrifuge tube
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Abstract
The invention relates to a method for extraction of total RNA from pomacea canaliculata muscular tissue. The method comprises the steps of: (1) flushing fresh pomacea canaliculata muscular tissue with DEPC water, putting the pomacea canaliculata muscular tissue into liquid nitrogen, grinding the pomacea canaliculata muscular tissue into fine powder under liquid nitrogen condition, and then transferring the powder into a centrifuge tube containing a cell lysis solution; (2) under ice bath condition, adding acidic water saturated phenol into the centrifuge tube of step (1), shaking the substances evenly, adding sodium acetate, and a mixed solution of chloroform and isoamyl alcohol, and carrying out ice bath; (3) conducting centrifugation to separate a precipitate phase and a liquid phase; (4) transferring the liquid phase into a new centrifuge tube, and adding a mixed solution of chloroform and isoamyl alcohol; (5) performing centrifugation and taking the supernatant, adding an equal volume of isopropanol precipitate; (6) performing centrifugation, discarding the supernatant, adding 1-1.5mL of ethanol to wash the precipitate; and (7) repeating the step (6) 1-2 times, performing air drying, adding 50-100 microliters of RNA enzyme water to dissolve the precipitate, thus obtaining a pomacea canaliculata muscular tissue total RNA solution. The total RNA extracted by the method provided by the invention has the characteristics of high yield, high purity and good quality integrality, and can meet the application of molecular biology research.
Description
Technical field
The invention belongs to biology field, relate to the extraction of fresh water software biology total serum IgE, particularly relate to kind of the method extracting total serum IgE from Pomacea canaliculata muscular tissue.
Background technology
Total serum IgE (ribonucleic acid) separating and purifying technology is the basis of molecular biology research technology, from tissue, DNA purity is stable, integrity good, repeated high, the isolation and purification method of free of contamination total serum IgE, is the key determining subsequent experimental outcome quality.
Pomacea canaliculata has another name called Ampullaria gigas, Fructus Mali pumilae spiral shell, originates in South America Amazon River basin.Before and after 1980, because of its rich in protein, nutritional labeling is high and fertility is strong, and is introduced into Taiwan, Philippine and Japan as a kind of aquatic economic animal, and rapidly diffuse into East Asia and remaining country of Southeast Asia (
Halwart, 1994).Later due to the marketization failure, Pomacea canaliculata lost one's parents and rapidly diffusion result population outbreak, serious harm crop production (
Naylor, 1996).2000, World Conservation Union (World Conservation Union; International Union for
Conservation of Nature and Natural Resources;IUCN) the Invasive Alien Species Committee of Experts Pomacea canaliculata is classified as one of 100 kinds of pernicious Invasive Alien Species in the world (
Lowe et al., 2000), also it is wherein unique a kind of freshwater snails.In China mainland, Pomacea canaliculata was incorporated into Guangdong cultivation in 1981, in Guangdong after 1984, Guangxi, the ground such as Fujian starts extensively to cultivate, it is generalized to the ground such as Zhejiang, Jiangxi, Yunnan, Sichuan subsequently, the serious agricultural pests (Yu Xiao equality, 2001) of major part provinces and regions on the south the Changjiang river are become at present.2003, Pomacea canaliculata was listed in " blacklist " of 16 kinds of alien species of first batch of invasion China by State Environmental Protection Administration and the Chinese Academy of Sciences (2003).Outside traditional preventing and treating such as cultural control and physical control, from molecular level, how to disclose the invasion mechanism of Pomacea canaliculata, develop new controlling way and have become as the emphasis of Pomacea canaliculata research.
At present, the method for the most isolated and purified Pomacea canaliculata total tissue RNA is less, few with the Pomacea canaliculata total tissue RNA content of traditional method for extracting, and poor stability easily pollutes, it is impossible to meet follow-up requirement of experiment.Summary of the invention
In order to solve problem present in existing fresh water software biology Total RNAs extraction technology, it is an object of the invention to provide a kind of method extracting total serum IgE from Pomacea canaliculata muscular tissue, the molecular biology research for Pomacea canaliculata provides technical support and guarantee.
The present invention completes by the following technical programs:
(1) take fresh Pomacea canaliculata muscular tissue DEPC water to rinse 3-5 time, put into liquid nitrogen, under the conditions of liquid nitrogen, grind to form fine-powdered, be then transferred in the centrifuge tube containing 0.3 0.5mL cell pyrolysis liquid；
(2), under condition of ice bath, the centrifuge tube in step (1) adds acid water-saturated phenol, shakes up, add 0.1-0.2mL sodium acetate, 0.1-0.2mL chloroform and isoamyl alcohol mixed liquor, ice bath 15min；
(3) 4 DEG C are centrifuged, precipitation separation phase and liquid phase；
(4) transfer liquid phase is in new centrifuge tube, adds equal-volume chloroform, isoamyl alcohol mixed liquor；
(5) centrifugal, take supernatant, add equal-volume isopropanol-20 DEG C precipitation；
(6) centrifugal, abandon supernatant, add 1-1.5mL75% washing with alcohol precipitation；
(7) repeating step (6) 1-2 time, air is dried, and adds 50-100 μ L and precipitates without RNase water dissolution, obtains Pomacea canaliculata muscular tissue total rna solution.
Described cell pyrolysis liquid consists of: guanidinium isothiocyanate: 24 weight portions；Sodium lauryl sulphate 0.4 weight portion；Carbamide 0.4 weight portion；Sodium chloride 0.2 weight portion, adds 25 weight portion DEPC water, is settled to 50 volumes；Described water-saturated phenol usage amount is cell pyrolysis liquid 1/2；Chloroform in described chloroform, isoamyl alcohol mixed liquor: isoamyl alcohol is 24:1；The concentration of described sodium acetate solution is 3mol/l,
PH value is 5.2；Described chloroform, the addition of isoamyl alcohol mixed liquor are equal with the volume of cell pyrolysis liquid；Described centrifugal speed is 12000rpm.
The present invention is applicable to the RNA of fresh water software biological tissue in a small amount and extracts, and method is simply effective, is suitable for molecular biology research application.Specifically have the beneficial effect that: 1, operating procedure is simple, convenient.2, RNA yield is high；3, RNA purity is high.
Accompanying drawing explanation
Fig. 1 is carried total serum IgE electrophoretogram by the inventive method.
Fig. 2 is that tradition Trizol method extracts total serum IgE electrophoretogram.
Fig. 3 is template for extracting total serum IgE by the present invention, PCR electrophoretogram.
Detailed description of the invention
Below in conjunction with example, the present invention is described in further detail:
Reagent that embodiment is used and equipment
DEPC (pyrocarbonic acid diethyl ester) water: the DEPC adding 0.1% in distilled water shakes up rear ambient temperature overnight, then carries out autoclave sterilization.
Cell pyrolysis liquid: accurately weigh guanidinium isothiocyanate 24g, sodium lauryl sulphate
0.4g, carbamide 0.4g, sodium chloride 0.2g, add the DEPC water that processed of 25mL autoclave sterilization, then constant volume is to 50mL.
PH value is the 3mol/L sodium acetate solution of 5.2: anhydrous sodium acetate 12.42g, DEPC water 30mL dissolves, and glacial acetic acid adjusts pH value to 5.2, adds water and is settled to 50mL.
Chloroform: isoamyl alcohol mixed liquor: draw 24mL chloroform, adds the mixing of 1mL isoamyl alcohol.
75% ethanol: take 75mL dehydrated alcohol, adds DEPC water and i.e. obtains 75% ethanol to 100mL.
Embodiment
1
(1) take fresh Pomacea canaliculata muscular tissue 100mg DEPC water to rinse 3 or 5 times, put into liquid nitrogen, under the conditions of liquid nitrogen, grind to form fine-powdered, be then transferred in the centrifuge tube containing 0.5mL cell pyrolysis liquid；
(2) under condition of ice bath, the centrifuge tube in step (1) adds 0.25mL acidity water-saturated phenol, shakes up, add 0.05mL sodium acetate, then add 0.5mL chloroform, isoamyl alcohol mixed liquor, mixing, ice bath 15min;
(3) 4 DEG C, 12000rpm is centrifuged 10min, precipitation separation phase and liquid phase；
(4) transfer liquid phase is in new 1.5mL centrifuge tube, adds equal-volume chloroform, isoamyl alcohol mixed liquor；
(5) 4 DEG C, 12000rpm is centrifuged 10min, takes supernatant, is transferred to by supernatant in new 1.5mL centrifuge tube, adds equal-volume isopropanol-20 DEG C precipitation 30min；
(6) 4 DEG C, 12000rpm is centrifuged 10min, abandons supernatant, and precipitation adds 1mL75% washing with alcohol precipitation；
(7) repeating step (6) 1 or 2 times, air is dried, and adds and precipitates without RNase water dissolution, obtains Pomacea canaliculata muscular tissue total rna solution.
In order to detect the quality of put forward total serum IgE, by the agarose gel denaturing formaldehyde electrophoresis detection of 1 %, find that the purity high (Fig. 1) of total serum IgE, integrity are preferable, and yield is high. the mRNA in total serum IgE is sufficient for Northern trace and the reaction such as hybridization analysis, cDNA synthesis, it can be seen that the RNA extracted does not has the pollution of RNA from electrophoretogram, the ratio seeing 28S Yu 18S that can also will be apparent from is about 2: 1, shows do not have RNA to degrade, and purity is high.
Embodiment
2
The comparison of various experimental techniques:
See table
As can be seen from the above table, the ratio of OD260/OD280 and OD260/OD230 of the RNA that RNA extraction method of the present invention is extracted the most in the preferred range, and has certain gap with the ratio of OD260/OD280 and OD260/OD230 of the RNA of the fresh water software biology Pomacea canaliculata of traditional Trizol extraction method extraction with standard value.This explanation, the RNA mass that the inventive method obtains is higher, and purity, integrity degree are more preferable, can fully meet follow-up molecular biology experiment.
Embodiment
3
Always
RNA
Reverse transcription and
TSP
The amplification of gene
The cDNA Synthesis Kit using the raw work biology in Shanghai to provide carries out reverse transcription.The Pomacea canaliculata muscular tissue total serum IgE extracted by the present invention is template, utilizes random primer to synthesize cDNA. under the effect of reverse transcription and then carries out PCR reaction, and reaction condition is as follows: 94 ° of C denaturations 3min；Then 94 ° of C degeneration 30s, 55 ° of C annealing 30s, 72 ° of C extend 1min, totally 35 circulations；Last 72 ° of C extend 10min.The PCR product of gained all with 1.0% agarose gel electrophoresis analysis.
Reaction system is as follows:
10×PCR
Buffer
5μl
dNTP Mixture (each
2.5mM)
4μl
Template
DNA
2μl
Primer F (10μM)
1μl
Primer R (10μM)
1μl
TaKaRa Taq (5U/μl)
0.25μl
Add ddH2O Up to 50μl
Electrophoresis result such as Fig. 3, it is thus achieved that the target fragment about total length 2300bp.Therefore, the total serum IgE extracted by the inventive method succeeds.
Claims (8)
1. the method extracting total serum IgE from Pomacea canaliculata muscular tissue, it is characterised in that comprise the following steps:
(1) take fresh Pomacea canaliculata muscular tissue DEPC water to rinse 3-5 time, put into liquid nitrogen, under the conditions of liquid nitrogen, grind to form fine-powdered, be then transferred in the centrifuge tube containing 0.3 0.5mL cell pyrolysis liquid；
(2), under condition of ice bath, the centrifuge tube in step (1) adds acid water-saturated phenol, shakes up, add 0.1-0.2mL sodium acetate, 0.1-0.2mL chloroform and isoamyl alcohol mixed liquor, ice bath 15min；
(3) 4 DEG C are centrifuged, precipitation separation phase and liquid phase；
(4) transfer liquid phase is in new centrifuge tube, adds equal-volume chloroform, isoamyl alcohol mixed liquor；
(5) centrifugal, take supernatant, add equal-volume isopropanol-20 DEG C precipitation；
(6) centrifugal, abandon supernatant, add 1-1.5mL75% washing with alcohol precipitation；
(7) repeating step (6) 1-2 time, air is dried, and adds 50-100 μ L and precipitates without RNase water dissolution, obtains Pomacea canaliculata muscular tissue total rna solution.
Method the most according to claim 1, it is characterised in that described cell pyrolysis liquid consists of:
Guanidinium isothiocyanate
24 weight portions,
Sodium lauryl sulphate
0.4 weight portion,
Carbamide
0.4 weight portion,
Sodium chloride
0.2 weight portion,
Add 25 weight portion DEPC water, be settled to 50 volumes.
Method the most according to claim 1, it is characterised in that described water-saturated phenol usage amount is cell pyrolysis liquid 1/2.
Method the most according to claim 1, it is characterised in that the chloroform in described chloroform and isoamyl alcohol mixed liquor: isoamyl alcohol is 24:1.
Method the most according to claim 1, it is characterised in that the concentration of described sodium acetate solution is 3mol/l, pH value is 5.2.
6. according to the method described in claim 1 or 3, it is characterised in that the addition of described chloroform and isoamyl alcohol mixed liquor is equal with the volume of cell pyrolysis liquid.
Method the most according to claim 1, it is characterised in that described centrifugal speed is 12000rpm.
Method the most according to claim 1, it is characterised in that described DEPC is pyrocarbonic acid diethyl ester.
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|---|---|---|---|
| CN201510021161.3A CN105838707A (en) | 2015-01-16 | 2015-01-16 | Method for extraction of total RNA from pomacea canaliculata muscular tissue |
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| CN201510021161.3A CN105838707A (en) | 2015-01-16 | 2015-01-16 | Method for extraction of total RNA from pomacea canaliculata muscular tissue |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107523564A (en) * | 2017-10-16 | 2017-12-29 | 皖南医学院 | A kind of extracting method of simple economy oncomelania total tissue RNA |
| CN109055387A (en) * | 2018-08-28 | 2018-12-21 | 中国计量大学 | The protein and its cloning process of Pomacea canaliculata cold tolerance related gene FAD9, its coding |
-
2015
- 2015-01-16 CN CN201510021161.3A patent/CN105838707A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107523564A (en) * | 2017-10-16 | 2017-12-29 | 皖南医学院 | A kind of extracting method of simple economy oncomelania total tissue RNA |
| CN109055387A (en) * | 2018-08-28 | 2018-12-21 | 中国计量大学 | The protein and its cloning process of Pomacea canaliculata cold tolerance related gene FAD9, its coding |
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Application publication date: 20160810 |