CN102864140A - Micro-blood RNA (ribonucleic acid) quick separation method through nucleic acid adsorption - Google Patents
Micro-blood RNA (ribonucleic acid) quick separation method through nucleic acid adsorption Download PDFInfo
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- CN102864140A CN102864140A CN2012103327552A CN201210332755A CN102864140A CN 102864140 A CN102864140 A CN 102864140A CN 2012103327552 A CN2012103327552 A CN 2012103327552A CN 201210332755 A CN201210332755 A CN 201210332755A CN 102864140 A CN102864140 A CN 102864140A
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Abstract
The invention discloses a micro-blood RNA (ribonucleic acid) quick separation method through nucleic acid adsorption. A nucleic acid adsorption centrifuge shield and RNA extraction reagent are used for quickly separating RNA from micro-blood, wherein the RNA extraction reagent consists of red blood cell lysis solution, dissociation solution, removing solution, washing solution and separation solution. The micro-blood RNA quick separation method through nucleic acid adsorption has the advantages that the method is simple and convenient to operate, the time is short, the sampled quantity is small, the purity of the extracted RNA is high, the RNA is not polluted by protein and DNA (deoxyribonucleic acid), the superiority to the traditional blood RNA extraction method is shown, and application values of clinical popularization of the method for disease diagnoses and researches are further represented.
Description
Technical field
The invention belongs to blood rna separation method in the technical field of molecular biology, be specifically related to rapid isolation of RNA from micro blood, adopt specific RNA separation agent and nucleic acid absorption centrifuge shield combined utilization, RNA in the energy sharp separation blood, its isolated RNA output and purity are high, pollute without protein and genomic dna, easy and simple to handle, consuming time short.
Background technology
At present, for the extraction of RNA in the blood, adopt lymphocyte separation medium to isolate first mononuclearcell both at home and abroad more, then carry out cracking and extract.Need a large amount of blood because separate mononuclearcell, brought difficulty and inconvenient to sampling like this, more importantly bring unnecessary misery and psychological burden to patient, and impact and limited clinical detection diagnosis and research work to disease.In addition, owing to containing a large amount of RNA enzymes and some composition in the red corpuscle in the blood, if process and totally can not have a strong impact on the extraction quality of RNA and the success of subsequent experimental, traditional extraction RNA method comprises uses lymphocyte separation medium to be difficult to accomplish this point, and length consuming time, complex operation.This also is to affect when RNA extracts in the blood easily to degrade and yield poorly and impure major reason.
Summary of the invention
Deficiency and influence factor for above-mentioned existence, the object of the invention is to, a kind of micro blood RNA nucleic acid absorption fast separating process is provided, establishment endogenous RNA enzyme liberating RNA active and remove bib and composition, simple to operate quick, and purity is high, pollute without protein and genomic dna, and output is high.
In order to realize above-mentioned task, the present invention adopts following technical solution:
A kind of micro blood RNA nucleic acid absorption fast separating process, it is characterized in that, the method adopts nucleic acid absorption centrifuge shield, extract reagent rapid isolation of RNA from micro blood with RNA, described RNA extracts reagent by erythrocyte splitting solution, from separating solution, removes solution, washings is molten to be formed with separation solution, specifically by operating process is:
At first remove red corpuscle and fragment in the micro blood by the cracking of erythrocyte splitting solution, using from separating solution separates the RNA in the mononuclearcell again, the activity that suppresses simultaneously endogenous RNA enzyme liberating RNA, its solution is adsorbed RNA by adsorption layer in the nucleic acid adsorption tube, with removing solution removal protein and other impurity components, clean with washing soln again, with separation solution RNA and nucleic acid adsorption layer are broken away from last, thereby obtain ultrapure RNA;
Described RNA extracts each component of reagent and material concentration (mol/L) composition by the final concentration volume is:
The NH of erythrocyte splitting solution: 0.01-1mol/L
4Cl, the KHCO of 0.0001-0.3mol/L
3, 0.00001-1mol/L EDTA surplus is deionized water;
From the guanidinesalt of separating solution: 0.1-10mol/L, the C of 0.005-0.5mol/L
6H
5Na
3O
7, the C of 0.0001-1mol/L
15H
28NaNO
3,, the C of 0.01-2mol/L
2H
6OS, the NaAc of 0.1-10mol/L, surplus is deionized water;
Remove the guanidinesalt of solution: 0.01-20mol/L, 0.001-0.60mol/L C
4H
11NO
3, surplus is the solution of ethanol and deionized water;
Washing soln: 0.001-2mol/L NaCl, 0.0001-2mol/L C
4H
11NO
3, surplus is the solution of ethanol and deionized water,
Described nucleic acid absorption centrifuge shield comprises internal layer adsorption tube and outer collection tube, contains the adsorption medium layer in the internal layer adsorption tube, and outer layer sleeve is fluid collection tube, during use the internal layer adsorption tube is inserted in the outer collection tube.
Micro blood RNA nucleic acid absorption fast separating process of the present invention, easy and simple to handle, consuming time short, sampling amount is few, the RNA purity of extracting is high, pollute without protein and DNA, show its tradition extract the advantage of blood rna method, more embody and have the clinical using value to medical diagnosis on disease and research of popularizing.
Description of drawings
Fig. 1 is experiment flow figure
Below in conjunction with drawings and Examples invention is further described.
Embodiment
According to technical scheme of the present invention, micro blood RNA nucleic acid absorption fast separating process of the present invention, whole time consumption of experimental process is short, and energy establishment RNA enzymic activity, add the specific adsorption effect, so that the RNA that extracts does not degrade, activity is high, purity is high.
Nucleic acid absorption centrifuge shield comprises internal layer adsorption tube and outer collection tube, wherein, contain the adsorption medium layer in the internal layer adsorption tube, outer layer sleeve is fluid collection tube, needs the internal layer adsorption tube is inserted in the outer collection tube when whenever adding a reagent, discards waste liquid in the collection tube after centrifugal, again adsorption tube is returned in the collection tube, when adding parting liquid at last, need adsorption tube is inserted in the collection tube of clean (new), pass through again the RNA of centrifugal acquisition purifying.
The nucleic acid sorbing material is circular paper, and RNA extracts reagent by erythrocyte splitting solution, from separating solution, removes solution, and washings is molten to be formed with separation solution.
RNA extracts each component of reagent and material concentration (mol/L) composition by the final concentration volume is:
The NH of erythrocyte splitting solution: 0.01-1mol/L
4Cl, the KHCO of 0.0001-0.3mol/L
3, 0.00001-1mol/L EDTA, surplus is deionized water;
From the guanidinesalt of separating solution: 0.1-10mol/L, the C of 0.005-0.5mol/L
6H
5Na
3O
7, the C of 0.0001-1mol/L
15H
28NaNO
3,, the C of 0.01-2mol/L
2H
6OS, the NaAc of 0.1-10mol/L, surplus is deionized water;
Remove the guanidinesalt of solution: 0.01-20mol/L, 0.001-0.60mol/L C
4H
11NO
3, surplus is the mixing solutions (ethanol: deionized water=2:1, v/v) of ethanol and deionized water;
Washing soln: 0.001-2mol/L NaCl, 0.0001-2mol/L C
4H
11NO
3, surplus is the mixing solutions (ethanol: deionized water=2:1, v/v) of ethanol and deionized water.
Below be the specific embodiment that the contriver provides, operating process is referring to shown in Figure 1:
1, get 0.5-1ml blood, add 3 times of volume erythrocyte splitting solution mixings, room temperature is placed 5min.
2, the centrifugal 1min supernatant discarded of 4000r;
3, add 1ml in the precipitation from separating the solution mixing, room temperature is placed 5min.
4, liquid is sucked in the nucleic acid adsorption tube, room temperature is placed 2min.
5, the centrifugal 30s of 12000r discards the waste liquid in the collection tube, and adsorption tube is turned back in the collection tube.
6, add removing solution, the centrifugal 30s of 12000r discards the waste liquid in the collection tube, and adsorption tube is turned back in the collection tube.
7, add washing soln, 12000r, centrifugal 30s discards the waste liquid in the collection tube.
8, adsorption tube is inserted in the clean centrifuge tube, after bonus point exsolution liquid chamber temperature was placed 2min, the centrifugal 1min of 12000r namely obtained the RNA of purifying.
Claims (2)
1. a micro blood RNA nucleic acid adsorbs fast separating process, it is characterized in that, the method adopts nucleic acid absorption centrifuge shield, extract reagent rapid isolation of RNA from micro blood with RNA, described RNA extracts reagent by erythrocyte splitting solution, from separating solution, removes solution, washings is molten to be formed with separation solution, specifically by operation steps is:
At first remove red corpuscle and fragment in the micro blood by the cracking of erythrocyte splitting solution, using from separating solution separates the RNA in the mononuclearcell again, the activity that suppresses simultaneously endogenous RNA enzyme liberating RNA, its solution is adsorbed RNA by adsorption layer in the nucleic acid adsorption tube, with removing solution removal protein and other impurity components, clean with washing soln again, with separation solution RNA and nucleic acid adsorption layer are broken away from last, thereby obtain ultrapure RNA;
Described RNA extracts each component of reagent and material concentration (mol/L) composition by the final concentration volume is:
The NH of erythrocyte splitting solution: 0.01-1mol/L
4Cl, the KHCO of 0.0001-0.3mol/L
3, 0.00001-1mol/L EDTA surplus is deionized water;
From the guanidinesalt of separating solution: 0.1-10mol/L, the C of 0.005-0.5mol/L
6H
5Na
3O
7, the C of 0.0001-1mol/L
15H
28NaNO
3,, the C of 0.01-2mol/L
2H
6OS, the NaAc of 0.1-10mol/L, surplus is deionized water;
Remove the guanidinesalt of solution: 0.01-20mol/L, 0.001-0.60mol/L C
4H
11NO
3, surplus is the mixing solutions of ethanol and deionized water;
Washing soln: 0.001-2mol/L NaCl, 0.0001-2mol/L C
4H
11NO
3, surplus is the mixing solutions of ethanol and deionized water.
2. the method for claim 1 is characterized in that, described nucleic acid absorption centrifuge shield comprises internal layer adsorption tube and outer collection tube, wherein, contain the adsorption medium layer in the internal layer adsorption tube, outer layer sleeve is fluid collection tube, during use the internal layer adsorption tube is inserted in the outer collection tube.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103160496A (en) * | 2013-04-02 | 2013-06-19 | 中国农业科学院北京畜牧兽医研究所 | Method for extracting and purifying total RNA (ribonucleic acid) of blood of large-scale experimental animal |
| CN108277219A (en) * | 2018-04-24 | 2018-07-13 | 青岛瑞思德生物科技有限公司 | A kind of reagent and extracting method of blood sample RNA extractions |
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| CN1922318A (en) * | 2003-12-24 | 2007-02-28 | 3M创新有限公司 | Methods for nucleic acid isolation and kits using solid phase material |
| CN101413018A (en) * | 2008-12-09 | 2009-04-22 | 中南大学 | Method for extracting genome DNA |
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| CN1922318A (en) * | 2003-12-24 | 2007-02-28 | 3M创新有限公司 | Methods for nucleic acid isolation and kits using solid phase material |
| CN101413018A (en) * | 2008-12-09 | 2009-04-22 | 中南大学 | Method for extracting genome DNA |
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| 林明等: "从血液中提取总RNA的一种快速高效方法", 《中国生物化学与分子生物学报》 * |
| 饶国洲 等: "高纯化质粒DNA分离方法", 《现代检验医学杂志》 * |
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| 黄留玉 主编: "《PCR最新技术原理、方法及应用》", 31 January 2005, 化学工业出版社 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103160496A (en) * | 2013-04-02 | 2013-06-19 | 中国农业科学院北京畜牧兽医研究所 | Method for extracting and purifying total RNA (ribonucleic acid) of blood of large-scale experimental animal |
| CN103160496B (en) * | 2013-04-02 | 2014-12-24 | 中国农业科学院北京畜牧兽医研究所 | Method for extracting and purifying total RNA (ribonucleic acid) of blood of large-scale experimental animal |
| CN108277219A (en) * | 2018-04-24 | 2018-07-13 | 青岛瑞思德生物科技有限公司 | A kind of reagent and extracting method of blood sample RNA extractions |
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