CN103642793A - Method for extracting jian carp muscle RNA for sequencing transcriptome - Google Patents
Method for extracting jian carp muscle RNA for sequencing transcriptome Download PDFInfo
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- CN103642793A CN103642793A CN201310535183.2A CN201310535183A CN103642793A CN 103642793 A CN103642793 A CN 103642793A CN 201310535183 A CN201310535183 A CN 201310535183A CN 103642793 A CN103642793 A CN 103642793A
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Abstract
The invention relates to a method for extracting jian carp muscle RNA for sequencing transcriptome, and especially relates to a method for extracting jian carp muscle RNA for sequencing transcriptome with high purity, good integrity and high yield, and the invention belongs to the field of molecular biology technology. The invention comprises the following steps: grinding jian carp muscular tissues in liquid nitrogen into powder; adding a denaturing solution and homogenizing; adding RNAse-Free water which contains protease K; oscillating and mixing; centrifuging and fetching a supernatant; adding chloroform and mixing; centrifuging and fetching a supernatant; and then adding a high-salt solution and isopropanol and obtaining a deposition; washing with ethanol and drying. The muscle RNA extracted by the invention has the advantages of high yield, high purity and good integrity, and can be used for sequencing transcriptome.
Description
Technical field
The present invention relates to a kind ofly for transcribing the extracting method of the jian carp muscle RNA of group order-checking, especially a kind of DNA purity is high, integrity good, yield is high for transcribing the method for the jian carp muscle RNA of group order-checking, belongs to technical field of molecular biology.
Background technology
The summation of transcribing group and be all RNA that certain species particular organization produces under a certain functional status is important means for research cell phenotype and function.Transcribing group order-checking (RNA-Seq) and refer to and utilize s-generation high throughput sequencing technologies to carry out cDNA order-checking, obtain rapidly the nearly all transcript of a certain species under a certain state comprehensively, is that the powerful tool of organizing complicacy is transcribed in further investigation at present.And have relatively high expectations at purity, integrity, concentration three aspects: for transcribing the RNA of group order-checking, so that the extraction of RNA just seems is particularly important, be the committed step of transcribing group order-checking.
Jian carp muscle tissue water content approaches 70%, protein content reaches 18%, be rich in collagen protein, mucopolysaccharide, but cell content is few, organize rna content low, adopt conventional RNA extracting method to be difficult to complete homogenate, and the product obtaining often contains the gluey insolubless such as mucopolysaccharide, yield is low, purity is not high, integrity is poor, do not reach for transcribing the requirement of the RNA quality of group order-checking, so that follow-up experiment cannot be carried out.
Summary of the invention
The object of this invention is to provide a kind ofly for transcribing the extracting method of the jian carp muscle RNA of group order-checking, it is applicable to the total RNA of muscle and extracts, and the muscle RNA yield that adopts the inventive method to extract is high, and purity is high, integrity is good, can be for transcribing group order-checking.Technical scheme is as follows:
For transcribing an extracting method of the jian carp muscle RNA of group order-checking, comprise the steps:
(1). get jian carp muscle tissue 10 ~ 50mg, drop into and be equipped with in the mortar of liquid nitrogen, limit adds liquid nitrogen limit grinds, and tissue is ground to Powdered;
(2) by powder transfer to centrifuge tube A, to it, add 400～600 μ l sex change liquid, with refiner at homogenate 10 ~ 40s on ice;
(3) in centrifuge tube A, add 550～650 μ l DEPC water, vibration mixes; Then the water that adds the RNAse-Free that 5～15 μ l contain Proteinase K, vibration mixes; Water-bath 8 ~ 12min at 50～60 ℃, needs the centrifuge tube that turns upside down therebetween again;
(4) the centrifugal 3～7min of 12000～14000 r/min under room temperature, shifts supernatant liquor in centrifuge tube B;
(5) in centrifuge tube B, add 150～250 μ l chloroforms, shake 15 ~ 20s, the standing 3 ~ 5min of room temperature; At the temperature of 4 ℃, with the centrifugal 8～12min of 12000～14000 r/min, shift supernatant liquor in centrifuge tube C;
(6) in centrifuge tube C, add 200～300 μ l high level salt solutions and 200～300 μ l Virahols, mix, room temperature is placed 8 ~ 12min, and described high level salt solution includes 0.8mol/L trisodium citrate and 1.2mol/L sodium-chlor, and solvent is water;
At (7) 4 ℃ of temperature, with the centrifugal 10 ~ 20min of 12000～14000 r/min, abandon supernatant liquor;
(8) precipitation is centrifugal 2 ~ 4 min of 70～80% aqueous ethanolic solution washing twice, 7000 ~ 8000 rpm/min by volumetric concentration, supernatant discarded;
(9) drying at room temperature precipitation is 2 ~ 5 minutes, adds 15 ~ 30 μ l DEPC water dissolution RNA, and-70 ~-90 ℃ of degree are preserved.
The composition and ratio of described sex change liquid can be:
Guanidinium isothiocyanate 23.6320g, trisodium citrate 0.3676g, sarcosyl 0.25g, beta-mercaptoethanol 1ml, all the other are deionized water, total amount 50ml.After conversion, that is: guanidinium isothiocyanate 472.64 mg/ml, trisodium citrate 7.252 mg/ml, sarcosyl 5 mg/ml, beta-mercaptoethanol 20 μ l/ml, solvent is water.
The mass concentration of described DEPC water can be 0.05～0.15%;
In the water of the described RNAse-Free that contains Proteinase K, the mass concentration of Proteinase K is 20mg/ml;
The principle that mainly realizes of the present invention: utilize high level salt solution to remove the jellies such as mucopolysaccharide.First use liquid nitrogen grinding, avoid RNA degraded, the homogenate of recycling refiner, homogenate completely.Adding Proteinase K is mainly the albumen such as digestion collagen protein and nucleoprotein.This method tissue mass, sex change liquid measure ratio are suitable, and homogenate is abundant, avoid RNA degraded, can extract the RNA in muscle.
beneficial effect
The muscle RNA purity that adopts the inventive method to extract is high, integrity good, can be for transcribing group order-checking.
Accompanying drawing explanation
Fig. 1 is that embodiment 1 extracts the also Agilent 2100 detection figure of the muscle RNA of purifying
Fig. 2 is RNA agarose electrophoresis figure in reference examples 1
Embodiment
embodiment 1
1. get jian carp muscle tissue 30 mg, drop into and be equipped with in the mortar of liquid nitrogen immediately, limit adds liquid nitrogen limit grinds, and tissue is ground to Powdered.
By powder transfer to centrifuge tube A, to it, add 500 μ l sex change liquid, with refiner at homogenate 30s on ice.
3. in centrifuge tube A, add 590 μ l DEPC water (0.1%w/w) dilution homogenates, vibration mixes.Then the water that adds the RNAse-Free that 10 μ l contain Proteinase K, the concentration of Proteinase K is 20mg/ml, vibration mixes.55 ℃ of water-bath 10min, centrifuge tube twice therebetween turns upside down.
4. the centrifugal 5min of 13000r/min under room temperature, shifts supernatant in centrifuge tube B.
5. in centrifuge tube B, add 200 μ l chloroforms, acutely shake 18s, the standing 4min of room temperature.At 4 ℃ of temperature, with the centrifugal 10min of 13000r/min, shift supernatant in centrifuge tube C.
6. in centrifuge tube C, add 250 μ l high level salt solutions (contain 0.8mol/L trisodium citrate and 1.2mol/L sodium-chlor, solvent is water) and 250 μ l Virahols, mix, room temperature is placed 10min.
7. at 4 ℃ of temperature, use the centrifugal 15min of 13000r/min, abandon supernatant.
8. precipitation is centrifugal 3 min of 75% aqueous ethanolic solution washing twice, 7500 rpm/min by volumetric concentration, careful supernatant discarded.
9. drying at room temperature precipitation is 4 minutes, adds 20 μ l DEPC water dissolution RNA, and-80 ℃ of degree are preserved.
The composition and ratio of sex change liquid is: guanidinium isothiocyanate 23.6320g, trisodium citrate 0.3676g, sarcosyl 0.25g, beta-mercaptoethanol 1ml, all the other are deionized water, total amount 50ml.
DEPC quality concentration is 0.1%.
After testing, in the present embodiment, extract the OD of the RNA obtaining
260/280value, between 1.98 ± 0.04, illustrates that sample purity is good.In addition, by Aglient Technologies 2100 Bioanalyzer, detect, as Fig. 1, RNA concentration reaches 207 ng/ μ l, and RIN value is 8.5, illustrates that the RNA sample quality extracting is qualified, integrity degree is good, and total amount and concentration meet the more than twice and twice group order-checking experiment demand of transcribing.
reference examples 1
Traditional RNA extracting method:
1. get jian carp muscle tissue 50 mg, insert in 1.5ml centrifuge tube.To it, add 1ml Trizol sex change liquid, with refiner homogenate 30s.
2. in centrifuge tube, add 200 μ l chloroforms, vibration 15s, standing 2min.
3. 4 ℃, the centrifugal 15min of 12000r/min, gets supernatant.
6. in supernatant, add 500 μ l Virahols, mix, room temperature is placed 10min.
7. again at the temperature of 4 ℃, the centrifugal 10min of 12000r/min, abandons supernatant.
8. adding 1ml volumetric concentration is 75% aqueous ethanolic solution, washing precipitation.Centrifugal 3 min of 7500 rpm/min, abandon supernatant.
9. dry, drying at room temperature precipitation 4 minutes, adds 20 μ l DEPC water dissolution RNA, and-80 ℃ of degree are preserved.
After testing, in the present embodiment, extract the OD of the RNA obtaining
260/280value, between 1.6 ± 0.08, illustrates that RNA purity is not high.In addition, from agarose electrophoresis figure (Fig. 2), bar, with disperse shape, illustrates that RNA has degraded.In a word, the jian carp muscle RNA quality of extracting by conventional Trizol method does not reach the requirement of transcribing group order-checking.
reference examples 2
Be by high level salt solution, centrifuged supernatant not to be removed the operation of the jellies such as mucopolysaccharide with the difference of embodiment 1, particularly, the step of this reference examples is as follows:
1. get jian carp muscle tissue 30 mg, drop into and be equipped with in the mortar of liquid nitrogen immediately, limit adds liquid nitrogen limit grinds, and tissue is ground to Powdered.
By powder transfer to centrifuge tube A, to it, add 500 μ l sex change liquid, with refiner at homogenate 30s on ice.
3. in centrifuge tube A, add 590 μ l DEPC water (0.1%w/w) dilution homogenates, vibration mixes.Then the water that adds the RNAse-Free that 10 μ l contain Proteinase K, the concentration of Proteinase K is 20mg/ml, vibration mixes.55 ℃ of water-bath 10min, centrifuge tube twice therebetween turns upside down.
4. the centrifugal 5min of 13000r/min under room temperature, shifts supernatant in centrifuge tube B.
5. in centrifuge tube B, add 200 μ l chloroforms, acutely shake 18s, the standing 4min of room temperature.At 4 ℃ of temperature, with the centrifugal 10min of 13000r/min, shift supernatant in centrifuge tube C.
6. in centrifuge tube C, add 250 μ l Virahols, mix, room temperature is placed 10min.
7. at 4 ℃ of temperature, use the centrifugal 15min of 13000r/min, abandon supernatant.
8. precipitation is centrifugal 3 min of 75% aqueous ethanolic solution washing twice, 7500 rpm/min by volumetric concentration, careful supernatant discarded.
9. drying at room temperature precipitation is 4 minutes, adds 20 μ l DEPC water dissolution RNA, and-80 ℃ of degree are preserved.
The composition and ratio of sex change liquid is: guanidinium isothiocyanate 23.6320g, trisodium citrate 0.3676g, sarcosyl 0.25g, beta-mercaptoethanol 1ml, total amount 50ml, surplus is deionized water.
DEPC quality concentration is 0.1%.
After testing, in the present embodiment, extract the OD of the RNA obtaining
260/280value, between 1.65 ± 0.08, illustrates that sample purity is not high.By Aglient Technologies 2100 Bioanalyzer, detect, RNA concentration is about 112 ng/ μ l, and RIN value is 6.0, illustrates that the RNA sample quality and the integrity degree that extract are all comparatively general.
reference examples 3
Be with the difference of embodiment 1 operation of by Proteinase K, collagen protein etc. not being processed, particularly, the step of this reference examples is as follows:
1. get jian carp muscle tissue 30 mg, drop into and be equipped with in the mortar of liquid nitrogen immediately, limit adds liquid nitrogen limit grinds, and tissue is ground to Powdered.
By powder transfer to centrifuge tube A, to it, add 500 μ l sex change liquid, with refiner at homogenate 30s on ice.
3. in centrifuge tube A, add 590 μ l DEPC water (0.1%w/w) dilution homogenates, vibration mixes.
4. the centrifugal 5min of 13000r/min under room temperature, shifts supernatant in centrifuge tube B.
5. in centrifuge tube B, add 200 μ l chloroforms, acutely shake 18s, the standing 4min of room temperature.At 4 ℃ of temperature, with the centrifugal 10min of 13000r/min, shift supernatant in centrifuge tube C.
6. in centrifuge tube C, add 250 μ l high level salt solutions (contain 0.8mol/L trisodium citrate and 1.2mol/L sodium-chlor, solvent is water) and 250 μ l Virahols, mix, room temperature is placed 10min.
7. at 4 ℃ of temperature, use the centrifugal 15min of 13000r/min, abandon supernatant.
8. precipitation is centrifugal 3 min of 75% aqueous ethanolic solution washing twice, 7500 rpm/min by volumetric concentration, careful supernatant discarded.
9. drying at room temperature precipitation is 4 minutes, adds 20 μ l DEPC water dissolution RNA, and-80 ℃ of degree are preserved.
The composition and ratio of sex change liquid is: guanidinium isothiocyanate 23.6320g, trisodium citrate 0.3676g, sarcosyl 0.25g, beta-mercaptoethanol 1ml, total amount 50ml, surplus is deionized water.
DEPC quality concentration is 0.1%.
After testing, in the present embodiment, extract the OD of the RNA obtaining
260/280value, between 1.55 ± 0.04, illustrates that sample purity is not high.By Aglient Technologies 2100 Bioanalyzer, detect, RNA concentration is about 107 ng/ μ l, and RIN value is 5.5, illustrates that the RNA sample quality and the integrity degree that extract are all comparatively general.
Claims (4)
1. for transcribing an extracting method of the jian carp muscle RNA of group order-checking, it is characterized in that, comprise the steps:
(1) get jian carp muscle tissue 10 ~ 50mg, drop into and be equipped with in the mortar of liquid nitrogen, limit adds liquid nitrogen limit grinds, and tissue is ground to Powdered;
(2) by powder transfer to centrifuge tube A, to it, add 400～600 μ l sex change liquid, with refiner at homogenate 10 ~ 40s on ice;
(3) in centrifuge tube A, add 550～650 μ l DEPC water, vibration mixes; Then the water that adds the RNAse-Free that 5～15 μ l contain Proteinase K, vibration mixes; Water-bath 8 ~ 12min at 50～60 ℃, needs the centrifuge tube that turns upside down therebetween again;
(4) the centrifugal 3～7min of 12000～14000 r/min under room temperature, shifts supernatant liquor in centrifuge tube B;
(5) in centrifuge tube B, add 150～250 μ l chloroforms, acutely shake 15 ~ 20s, the standing 3 ~ 5min of room temperature; At the temperature of 4 ℃, with the centrifugal 8～12min of 12000～14000 r/min, shift supernatant liquor in centrifuge tube C;
(6) in centrifuge tube C, add 200～300 μ l high level salt solutions and 200～300 μ l Virahols, mix, room temperature is placed 8 ~ 12min, and described high level salt solution includes 0.8mol/L trisodium citrate and 1.2mol/L sodium-chlor, and solvent is water;
At (7) 4 ℃ of temperature, with the centrifugal 10 ~ 20min of 12000～14000 r/min, abandon supernatant liquor;
(8) precipitation is centrifugal 2 ~ 4 min of 70～80% aqueous ethanolic solution washing twice, 7000 ~ 8000 rpm/min by volumetric concentration, supernatant discarded;
(9) drying at room temperature precipitation is 2 ~ 5 minutes, adds 15 ~ 30 μ l DEPC water dissolution RNA, and-70 ~-90 ℃ of degree are preserved.
2. according to claim 1 for transcribing the extracting method of the jian carp muscle RNA of group order-checking, it is characterized in that: in described sex change liquid, include: guanidinium isothiocyanate 472.64 mg/ml, trisodium citrate 7.252 mg/ml, sarcosyl 5 mg/ml, beta-mercaptoethanol 20 μ l/ml, solvent is water.
3. according to claim 1 for transcribing the extracting method of the jian carp muscle RNA of group order-checking, it is characterized in that: the mass concentration of described DEPC water is 0.05～0.15%.
4. according to claim 1 for transcribing the extracting method of the jian carp muscle RNA of group order-checking, it is characterized in that: in the water of the described RNAse-Free that contains Proteinase K, the mass concentration of Proteinase K is 20mg/ml.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106282169A (en) * | 2016-11-01 | 2017-01-04 | 中国水产科学研究院淡水渔业研究中心 | A kind of rapid batch extracts the method for fish tissues total serum IgE |
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Patent Citations (5)
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| CN101165183A (en) * | 2006-10-19 | 2008-04-23 | 陕西西大北美基因股份有限公司 | Method for separating mRNA by using gold magnetism particles |
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| CN101914621A (en) * | 2010-08-18 | 2010-12-15 | 中国农业大学 | Method for mRNA quantitative detection of composition of porcine meat muscle fiber types |
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