CN110241112A - A kind of extracting method of adipose tissue RNA - Google Patents
A kind of extracting method of adipose tissue RNA Download PDFInfo
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Abstract
The present invention provides a kind of extracting methods of adipose tissue RNA, including adipose tissue to pre-process, extraction, Filter column adsorbs RNA, washing RNA, collection RNA and etc., wherein in Filter column absorption RNA step, by the way of DNaseI enzymic digestion gDNA is added dropwise on Filter column, the interference of gDNA is eliminated, while avoiding the damage of RNA, while the extracting method also reduces operating procedure duration, the risk for reducing RNA degradation improves the purity of RNA extraction.
Description
Technical field
The invention belongs to field of biotechnology, more particularly, to a kind of extracting method of adipose tissue RNA.
Background technique
Adipose tissue is the organ of energy storage, is important endocrine system, it affects the insulin sensitivity of body
Property, blood pressure level, endothelial function, fibrinolytic activity and inflammatory reaction, participate in a variety of important pathophysiological processes, also relate to
A variety of and obesity-related disease occurrence and development.We are studied rouge by the expression conditions for analysing in depth adipose tissue
The metabolic process of fat tissue analyzes it and all plays the role of to the regulation of pathologic, physiologic function vital, and studies these mistakes
The top priority of journey is to the adipose tissue RNA of stable acquisition high-purity.
The extracting method of RNA is one very mature technology of modern molecular biology, at present the existing RNA being much commercialized
Extracts kit.But different tissues can generate some special requirements, rouge due to the specificity of tissue in RNA extraction process
Fat organization unit volume cells number is less, and unit cell rna content is also relatively fewer, and which increase extract from adipose tissue
The difficulty of RNA.And since interfering substance is more, using conventional commercial kit, it is high-quality to tend not to stable acquisition
RNA is measured, a large amount of miRNA can be lost instead.
Summary of the invention
In view of this, the present invention is directed to propose a kind of extracting method of adipose tissue RNA, to solve adipose tissue RNA
It is difficult to effectively remove the impurity such as gDNA in extraction process, the problem of the acquisition high quality RNA of stability and high efficiency.
In order to achieve the above objectives, the technical scheme of the present invention is realized as follows:
A kind of extracting method of adipose tissue RNA, comprising the following steps:
(1) adipose tissue 0.5-250mg is taken, lysate is added, is homogenized with Syrup-homogenizing instrument to without obvious particle, is stored at room temperature
10min, then 10000g is centrifuged 5min, removes upper layer oil reservoir and lower layer's insoluble matter, takes middle layer spare；
(2) homogenate annex solution is added into step (1) taken middle layer, concussion mixes, then ice bath 10min；Phenol is added
Acid-chloroform, concussion mix, and 10000g is centrifuged 5min at room temperature, then take upper strata aqueous phase part, the anhydrous of 1.25 times of volumes is added
It is spare that mixed liquor is made in ethyl alcohol；
(3) mixed liquor prepared in step (2) is added drop-wise on Filter column, then 10000g is centrifuged 15s, discards waste liquid；
700 μ L cleaning solutions are added dropwise on Filter column again, 10000g is centrifuged 5-10s, discards waste liquid；80 μ L are finally added dropwise on Filter column film
DNase I enzyme mixation, is stored at room temperature 15min；
(4) 500 μ L cleaning solutions are added dropwise on the Filter column after standing, 10000g is centrifuged 5-10s, discards waste liquid, and repeat
This step；
(5) Filter column is centrifuged 1min in 10000g, discards waste liquid；The eluent of 95 DEG C of 100 μ L is added, is centrifuged 30s,
RNA is collected, and is stored in -80 DEG C.
Further, in step (1) Syrup-homogenizing instrument needed before use successively with DEPC water, 5% hydrogen peroxide, 75% ethyl alcohol and
DEPC water sterilization.
Further, in step (1) as taking adipose tissue be cryopreserved tissue, tissue should be washed away with PBS solution in advance
Remaining RNA saves liquid.
Further, after the imitative mixing centrifugation of phenol is added in step (2), if solution is not layered or layered effect is bad,
It then needs to be centrifuged again.
Further, the dehydrated alcohol being added in step (2) is dehydrated alcohol under room temperature.Further, step
(3) dehydrated alcohol is mixed in the washing solution being added in.
Further, each liquid being added dropwise in step (3) should come into full contact with entire Filter column film.
Further, dehydrated alcohol is mixed in the washing solution being added in step (4).
Further, step (3), (4), Filter column described in (5) are glass fibre membrane Filter column.
Compared with the existing technology, the extracting method of a kind of adipose tissue RNA of the present invention.It has the advantage that
(1) extracting method of a kind of adipose tissue RNA of the present invention, using DNase I enzyme on glass fibre membrane
GDNA is digested, remaining gDNA can have been effectively removed, while avoiding damage RNA.
(2) extracting method of a kind of adipose tissue RNA of the present invention, by DNase I enzymic digestion and RNA purification process
Merge, reduce operating procedure duration, reduce the risk of RNA degradation, improves the purity of RNA extraction.
In step (1), flesh tissue or cryopreserved tissue is can be used in adipose tissue, when using cryopreserved tissue, needs to mention
Before wash off the remaining RNA of tissue and save liquid, the cleaning method generally used is cleaned repeatedly with PBS solution, the purpose of cleaning
It is the interference for preventing residual RNAlater from extracting to RNA.After lysate is added, acutely to shake, it made sufficiently to act on RNA,
Then homogenized can make tissue sufficiently broken, stand 5min, to promote the release of miRNA, while to prevent organic extraction
When there is not stratified phenomenon, after homogenate is broken, by the way of centrifugation, remove the oil reservoir and precipitating in tissue.
Pregnant solution is added in step (2), promotes miRNA to be enriched with, ice bath 10min can accelerate the enrichment of miRNA, so
Addition phenolic acid-chloroform shakes mixing repeatedly and is extracted afterwards, and at this moment solution is in muddy shape, after centrifugation if without significantly dividing
Layer, then need to be centrifuged again, it is ensured that removal lipid pollution, until drawing upper strata aqueous phase to new centrifuge tube after having apparent layering
In.The dehydrated alcohol of 3 times of aqueous layer volumes is added, mixing is prepared into mixed solution, and dehydrated alcohol saves at room temperature, cannot
Use the dehydrated alcohol of cryo-conservation.
Nucleic acid is adsorbed using glass fibre membrane Filter column in step (3), mixed liquor is added drop-wise on adsorption column, with high salt dense
Under the conditions of degree, with the silanol group on glass fibre physical absorption can occur for nucleic acid.Cleaning solution and ethyl alcohol are added dropwise on Filter column
Mixed liquor can clean the impurity such as the albumen of Filter column surface attachment.Then DNase I is added dropwise on Filter column again, to digest
The DNA adsorbed by filter membrane, while the RNA molecule adsorbed on filter membrane being protected not damaged by DNase I digestion system, stand 15 points
Clock waits for that it completes digestion.In this course, the liquid being added dropwise on Filter column each time will ensure to fill with entire Filter column
Tap touching, it is unbonded to have prevented, it does not elute, it is indigested to happen.The step, will using the method for digesting DNA on column
RNA separation and DNA digestion combine, and save the reaction time, reduce reaction step, while avoiding introducing other salt ions
Equal impurity, it is most important that pollution and damage when DNA digestion can be effectively reduced in this step to RNA.
After step (4) is stood, cleaning solution is added dropwise on Filter column, washs the digestion product on Filter column, removes remaining
DNase I and salt ion.Complete RNA molecule is only remained on filter membrane at this time, avoids DNase I digestion body to greatest extent
It is the damage to RNA molecule, is mixed with dehydrated alcohol in cleaning solution, can remove the impurity such as albumen, then repeated washing is primary, reduces
Salt ionic concentration further purifies RNA, and the purity of RNA can be improved in this step.
When step (5) collects RNA, first sky is got rid of, and is removed residual liquid on Filter column, is contained salt ion in residual liquid,
The purity of RNA is influenced, the eluent of 95 DEG C of 100 μ L is added, can quickly dissolve RNA, improve the yield of RNA.
Detailed description of the invention
The attached drawing for constituting a part of the invention is used to provide further understanding of the present invention, schematic reality of the invention
It applies example and its explanation is used to explain the present invention, do not constitute improper limitations of the present invention.In the accompanying drawings:
Fig. 1 is that RNA described in the embodiment of the present invention 1 extracts electrophoretogram (wherein M is maker, and 1-3 is sample number 1-3).
Fig. 2 is that RNA described in comparative example 1 of the present invention extracts electrophoretogram (wherein M is maker, and 1-3 is sample number 1-3).
Fig. 3 is that RNA described in comparative example 2 of the present invention extracts electrophoretogram (wherein M is maker, and 1-3 is sample number 1-3).
Specific embodiment
It should be noted that in the absence of conflict, the feature in embodiment and embodiment in the present invention can phase
Mutually combination.
The extraction of the adipose tissue RNA of the different samples of embodiment 1.
One, sample and reagent information；
The adipose tissue of 3 kinds of samples is taken, sample ID is respectively as follows:
1. 1:-80 DEG C of number freezes in RNALater, 0.5mg；
2. number 2: fresh fat tissue, 250mg；
3. 3:-80 DEG C of number freezes in RNALater, 10mg；
Agents useful for same is commodity purchased product in embodiment, or by outsourcing products configuration；
Operating process
Two, extracting method
1. adipose tissue pre-processes:
1.1 are placed in the sample of number 1 and 3 in different centrifuge tubes, are separately added into appropriate PBS concussion cleaning, it is molten to discard PBS
Liquid operates several times repeatedly, it is ensured that washes away the remaining RNA of sample surface and saves liquid；The sample of number 2 is individually placed in centrifuge tube,
Without cleaning.
1.2 successively use Syrup-homogenizing instrument DEPC water, 5% hydrogen peroxide, 75% ethyl alcohol and DEPC water sterilization.
1.3 are separately added into the lysate (Lysis/Binding Buffer) of 900 μ L in 1.1 3 centrifuge tubes, acute
Violent shock is swung, be then homogenized with Syrup-homogenizing instrument to without obvious particle, be stored at room temperature 10min, and then 10000g is centrifuged 5min, is taken respectively
Interbed is into new centrifuge tube.
2. extraction
2.1 are separately added into the pregnant solution (miRNAHomogenate of 90 μ L in 1.3 described 3 new centrifuge tubes
Additive), oscillation mixes, and is subsequently placed in ice bath 10min on ice.
900 μ L acid phenol-chloroforms are added after 2.2 ice baths, concussion mixes 30-60sec, and room temperature 10000g is centrifuged 5min, draws
Upper strata aqueous phase is into new centrifuge tube.
3.RNA separation
3.1 are added the dehydrated alcohol of 1.25 times of volumes in upper strata aqueous phase, mix.
3.2 are put into Filter column in collecting pipe, and 3.1 obtained mixed liquors are added drop-wise on Filter column, 10,000 × g from
Heart 15sec abandons waste liquid, replaces collecting pipe.
3.3 are added dropwise 700 μ L cleaning solutions (cleaning solution is mixed with dehydrated alcohol) 10 on Filter column, and 000 × g is centrifuged 5-10sec,
Abandon waste liquid.
3.4 are added dropwise 80 μ L DNase I enzyme mixations (+70 μ LBuffer of 30 μ LDNase I proenzyme liquid) on Filter column,
It is stored at room temperature 15min.
3.5 are added dropwise 500 μ L cleaning solutions (cleaning solution is mixed with dehydrated alcohol) on Filter column, and 10,000 × g is centrifuged 5-
10sec abandons waste liquid.It is primary to repeat this step.
3.6 put back to Filter column in collecting pipe, 10,000 × g, and sky gets rid of 1min, remove rinsing liquid remaining in Filter column.
3.7 are put into Filter column in new collecting pipe, and 100 μ L are added and have been warmed up to 95 DEG C of eluent (Elution
Solution), it is centrifuged 30sec, collects RNA.
3.8 carry out RNA quantitative detection, and the results are shown in Table 1.
| Sample number | RNA concentration | 260/280 | 260/230 | RNA total amount/ug |
| 1 | 446.5 | 2.13 | 2.31 | 29.02 |
| 2 | 466.5 | 2.16 | 1.99 | 30.32 |
| 3 | 278.9 | 2.14 | 2.18 | 18.13 |
3.9 agarose gel electrophoresis detect RNA integrality.
Electrophoresis result is referring to Fig. 1.As can be seen from Figure, the effect after gDNA is digested on column.
Comparative example 1
Test is compared using sample used in embodiment:
1. adipose tissue pre-processes:
1.1 are placed in the sample of number 1 and 3 in different centrifuge tubes, are separately added into appropriate PBS concussion cleaning, it is molten to discard PBS
Liquid operates several times repeatedly, it is ensured that washes away the remaining RNA of sample surface and saves liquid；The sample of number 2 is individually placed in centrifuge tube,
Without cleaning.
1.2 successively use Syrup-homogenizing instrument DEPC water, 5% hydrogen peroxide, 75% ethyl alcohol and DEPC water sterilization.
1.3 are separately added into the lysate (Lysis/Binding Buffer) of 900 μ L in 1.1 3 centrifuge tubes, acute
Violent shock is swung, be then homogenized with Syrup-homogenizing instrument to without obvious particle, be stored at room temperature 10min, and then 10000g is centrifuged 5min, is taken respectively
Interbed is into new centrifuge tube.
2. extraction
2.1 are separately added into pregnant solution (the miRNA Homogenate of 90 μ L in 1.3 described 3 new centrifuge tubes
Additive), oscillation mixes, and is subsequently placed in ice bath 10min on ice.
900 μ L acid phenol-chloroforms are added after 2.2 ice baths, concussion mixes 30-60sec, and room temperature 10000g is centrifuged 5min, draws
Upper strata aqueous phase is into new centrifuge tube.
3.RNA separation
3.1 are added the dehydrated alcohol of 1.25 times of volumes in upper strata aqueous phase, mix.
3.2 are put into Filter column in collecting pipe, and 3.1 obtained mixed liquors are added in Filter column, 10,000 × g from
Heart 15sec abandons waste liquid, replaces collecting pipe.
3.3 are added dropwise 700 μ L cleaning solutions (cleaning solution is mixed with dehydrated alcohol) 10 on Filter column, and 000 × g is centrifuged 5-10sec,
Abandon waste liquid.
3.4 are added dropwise 500 μ L cleaning solutions (cleaning solution is mixed with dehydrated alcohol) on Filter column, and 10,000 × g is centrifuged 5-
10sec abandons waste liquid.It is primary to repeat this step.
3.5 put back to Filter column in collecting pipe, 10,000 × g, and sky gets rid of 1min, remove rinsing liquid remaining in Filter column.
3.6 are put into Filter column in new collecting pipe, and 100 μ L are added and have been warmed up to 95 DEG C of eluent (Elution
Solution), it is centrifuged 30sec, collects RNA.
3.7 80 μ L DNase I enzyme mixations (+70 μ of 30 μ LDNase I proenzyme liquid is added dropwise into collected RNA
LBuffer)。
3.8 carry out RNA quantitative detection, and the results are shown in Table 2.
| Sample number | RNA concentration | 260/280 | 260/230 | RNA total amount/ug |
| 1 | 97.4 | 2.2 | 1.9 | 6.98 |
| 2 | 31.6 | 2.1 | 0.65 | 2.05 |
| 3 | 16.1 | 2.2 | 0.1 | 1.05 |
3.9 agarose gel electrophoresis detect RNA integrality.
Electrophoresis result is referring to fig. 2.As can be seen from Figure, the effect after liquid phase digestion gDNA.
Comparative example 2
Test is compared using sample used in embodiment:
1. adipose tissue pre-processes:
1.1 are placed in the sample of number 1 and 3 in different centrifuge tubes, are separately added into appropriate PBS concussion cleaning, it is molten to discard PBS
Liquid operates several times repeatedly, it is ensured that washes away the remaining RNA of sample surface and saves liquid；The sample of number 2 is individually placed in centrifuge tube,
Without cleaning.
1.2 successively use Syrup-homogenizing instrument DEPC water, 5% hydrogen peroxide, 75% ethyl alcohol and DEPC water sterilization.
1.3 are separately added into the lysate (Lysis/Binding Buffer) of 900 μ L in 1.1 3 centrifuge tubes, acute
Violent shock is swung, be then homogenized with Syrup-homogenizing instrument to without obvious particle, be stored at room temperature 10min, and then 10000g is centrifuged 5min, is taken respectively
Interbed is into new centrifuge tube.
2. extraction
2.1 are separately added into pregnant solution (the miRNA Homogenate of 90 μ L in 1.3 described 3 new centrifuge tubes
Additive), oscillation mixes, and is subsequently placed in ice bath 10min on ice.
900 μ L acid phenol-chloroforms are added after 2.2 ice baths, concussion mixes 30-60sec, and room temperature 10000g is centrifuged 5min, draws
Upper strata aqueous phase is into new centrifuge tube.
3.RNA separation
3.1 are added the dehydrated alcohol of 1.25 times of volumes in upper strata aqueous phase, mix.
3.2 are put into Filter column in collecting pipe, and 3.1 obtained mixed liquors are added in Filter column, 10,000 × g from
Heart 15sec abandons waste liquid, replaces collecting pipe.
3.3 are added dropwise 700 μ L cleaning solutions (cleaning solution is mixed with dehydrated alcohol) 10 on Filter column, and 000 × g is centrifuged 5-10sec,
Abandon waste liquid.
3.4 are added dropwise 500 μ L cleaning solutions (cleaning solution is mixed with dehydrated alcohol) on Filter column, and 10,000 × g is centrifuged 5-
10sec abandons waste liquid.It is primary to repeat this step.
3.5 put back to Filter column in collecting pipe, 10,000 × g, and sky gets rid of 1min, remove rinsing liquid remaining in Filter column.
3.6 are put into Filter column in new collecting pipe, and 100 μ L are added and have been warmed up to 95 DEG C of eluent (Elution
Solution), it is centrifuged 30sec, collects RNA.
3.7 carry out RNA quantitative detection, and the results are shown in Table 3.
| Sample number | RNA concentration | 260/280 | 260/230 | RNA total amount/ug |
| 1 | 381.4 | 2.09 | 1.8 | 18.29 |
| 2 | 323.6 | 2.0 | 2 | 14.53 |
| 3 | 276.1 | 2.07 | 2.18 | 11.45 |
3.8 agarose gel electrophoresis detect RNA integrality.
Electrophoresis result is referring to Fig. 3, as can be seen from Figure, the effect of traditional extraction adipose tissue RNA.By 1 He of embodiment
From the point of view of result in comparative example 1-2, the extraction total amount that embodiment 1 digests the RNA after gDNA on column is apparently higher than in the liquid phase
GDNA is digested, is mixed with gDNA in the RNA after general extraction methods extraction, the total amount of RNA is caused to be lower than on column after digestion gDNA
RNA extraction total amount, and digest gDNA under liquid-phase condition and will cause the loss of RNA, influence the extraction total amount of RNA.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Within mind and principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (9)
1. a kind of extracting method of adipose tissue RNA, it is characterised in that the following steps are included:
(1) adipose tissue 0.5-250mg is taken, lysate is added, is homogenized with Syrup-homogenizing instrument to without obvious particle, is stored at room temperature 10min,
Then 10000g is centrifuged 5min, removes upper layer oil reservoir and lower layer's insoluble matter, takes middle layer spare；
(2) RNA pregnant solution is added into step (1) taken middle layer, concussion mixes, then ice bath 10min；Phenolic acid-chlorine is added
Imitative, concussion mixes, and 10000g is centrifuged 5min at room temperature, then takes upper strata aqueous phase part, the dehydrated alcohol of 1.25 times of volumes is added,
It is spare that mixed liquor is made；
(3) mixed liquor prepared in step (2) is added drop-wise on Filter column, then 10000g is centrifuged 15s, discards waste liquid；Exist again
700 μ L cleaning solutions are added dropwise on Filter column, 10000g is centrifuged 5-10s, discards waste liquid；80 μ L are finally added dropwise on Filter column film
DNaseI enzyme mixation, is stored at room temperature 15min；
(4) 500 μ L cleaning solutions are added dropwise on the Filter column after standing, 10000g is centrifuged 5-10s, discards waste liquid, duplicate of laying equal stress on step
Suddenly；
(5) Filter column is centrifuged 1min in 10000g, discards waste liquid；The eluent of 95 DEG C of 100 μ L is added, is centrifuged 30s, collects
RNA, and it is stored in -80 DEG C.
2. a kind of extracting method of adipose tissue RNA according to claim 1, it is characterised in that: Syrup-homogenizing instrument in step (1)
It is needed before use successively with DEPC water, 5% hydrogen peroxide, 75% ethyl alcohol and DEPC water cleaning and sterilizing.
3. a kind of extracting method of adipose tissue RNA according to claim 1, it is characterised in that: in step (1) as take
Adipose tissue is cryopreserved tissue, should wash away the remaining RNA of tissue with PBS solution in advance and save liquid.
4. a kind of extracting method of adipose tissue RNA according to claim 1, it is characterised in that: phenol is added in step (2)
After imitative mixing centrifugation, if solution is not layered or layered effect is bad, need to be centrifuged again.
5. a kind of extracting method of adipose tissue RNA according to claim 1, it is characterised in that: be added in step (2)
Dehydrated alcohol is dehydrated alcohol under room temperature.
6. a kind of extracting method of adipose tissue RNA according to claim 1, it is characterised in that: be added in step (3)
Dehydrated alcohol is mixed in washing solution.
7. a kind of extracting method of adipose tissue RNA according to claim 1, it is characterised in that: be added dropwise in step (3)
Each liquid should come into full contact with entire Filter column film.
8. a kind of extracting method of adipose tissue RNA according to claim 1, it is characterised in that: be added in step (4)
Dehydrated alcohol is mixed in washing solution.
9. a kind of extracting method of adipose tissue RNA according to claim 1, it is characterised in that: step (3), (4), (5)
Described in Filter column be glass fibre membrane Filter column.
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| CN103820433A (en) * | 2014-03-10 | 2014-05-28 | 北京诺禾致源生物信息科技有限公司 | Method for extracting mRNA (messenger ribose nucleic acid) from tatty tissue |
| CN109939222A (en) * | 2019-04-26 | 2019-06-28 | 中国人民解放军北部战区总医院 | CREG albumen is used to promote the medical usage of skeletal muscle regeneration |
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Application publication date: 20190917 |
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