CN112410402A - Free RNA precipitation aid - Google Patents
Free RNA precipitation aid Download PDFInfo
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- CN112410402A CN112410402A CN202011261598.1A CN202011261598A CN112410402A CN 112410402 A CN112410402 A CN 112410402A CN 202011261598 A CN202011261598 A CN 202011261598A CN 112410402 A CN112410402 A CN 112410402A
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- free rna
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- settling agent
- pcr
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
Abstract
The invention relates to a free RNA (ribonucleic acid) settling aid, wherein each 1ml of the settling aid comprises the following components: 5-10mg dextran sulfate, 0.01-0.02mg linear acrylamide and 0.1-0.2mmol acetate. The free RNA precipitation aid provided by the invention can accelerate precipitation in the cfRNA extraction process and improve the final extraction yield of the free RNA; in addition, the settling agent is convenient to use, can obviously improve the extraction rate of free RNA in a sample only by adding the settling agent into the existing RNA extraction kit, has no adverse effect on subsequent downstream molecular biology applications, such as RT-PCR, qRT-PCR and the like, and has wide application prospects.
Description
Technical Field
The invention belongs to the technical field of free RNA extraction, and particularly relates to a free RNA precipitation aid.
Background
RNA is a relatively unstable molecule that is easily degraded by ribonucleases. cfRNA includes RNA released in the circulation during apoptosis of human cells, mRNA, miRNAs, tRNAs, YRNA, circRNAs, piRNAs, rRNA, snRNA. Exogenous extracellular RNA from food and human microbiota has also been reported to enter the circulation and studies have demonstrated that cfRNA can serve as a biomarker for various diseases including cancer. Most patients have very low cfRNA content, which brings great challenges to subsequent detection.
Disclosure of Invention
The invention aims to provide a free RNA precipitation aid for accelerating precipitation in a cfRNA extraction process and improving the final extraction yield of cfRNA.
The invention provides a free RNA (ribonucleic acid) settling aid, wherein each 1ml of the settling aid comprises the following components: 5-10mg dextran sulfate, 0.01-0.02mg linear acrylamide and 0.1-0.2mmol acetate.
The acetate is ammonium acetate or potassium acetate.
The settling agent is stored in a test tube at the temperature of-10-20 ℃.
The invention also provides an application of the free RNA precipitation aid.
The settling agent is used for extracting free RNA of a body fluid sample.
The body fluid comprises plasma, serum, pleural fluid, ascites, cerebrospinal fluid or urine.
The settling agent is used for a sample extraction step of a molecular biology downstream test, such as RT-PCR or qRT-PCR and the like.
Advantageous effects
Compared with the prior art, the precipitation aid provided by the invention can effectively accelerate the precipitation of free RNA during the extraction of the free RNA of a body fluid sample, can obviously improve the extraction yield of the free RNA, and further can improve the detection rate of the biomarkers of the free RNA related diseases in the sample; in addition, the settling agent is convenient to use, can obviously improve the extraction rate of free RNA in a sample only by adding the settling agent into the conventional RNA extraction kit, and has wide application range.
Drawings
FIG. 1 shows the Ct value determination results in example 1.
FIG. 2 shows the Ct value determination results in example 2.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.
Example 1
The free RNA precipitation aid provided by the embodiment consists of the following components: 5mg/ml dextran sulfate, 0.01mg/ml linear acrylamide and 0.1mg/ml acetate.
Normal human serum sample is extracted by TRIzol reagent method cfRNA, and the effect of adding and not adding cfRNA settling agent is compared by using one-step method qRT-PCR method to detect housekeeping gene GAPDH CT value.
1. Nucleic acid extraction:
1) adding 0.5ml serum into 1ml TRIzol reagent, mixing, and standing at room temperature for 5-10 min.
2) 0.2ml of chloroform was added thereto, followed by vigorous shaking for 15 seconds and standing for 2 min.
3) Centrifuging at 4 deg.C for 12000g × 15min, and collecting supernatant.
4) Adding cfRNA precipitation aid with the addition of 100 mul, adding 0.5ml isopropanol, gently mixing the liquid in the tube, and standing at room temperature for 10 min. Meanwhile, only isopropanol was added without cfRNA precipitation aid in this step for comparison.
5) Centrifugation is carried out at 4 ℃ for 12000g × 10min, and the supernatant is discarded.
6) 1ml of 75% ethanol was added and the precipitate was washed gently. At 4 deg.C, 7500g × 5min, discard the supernatant.
7) Air drying, adding 30 μ l DEPC H2Dissolving O (promoting dissolution at 65 ℃ for 10-15 min).
8) Stored at-70 ℃ or used directly in the next experiment.
2. And (3) detection:
and (3) carrying out fluorescent quantitative PCR verification on the obtained cfRNA by using a housekeeping gene GAPDH, and judging a detection result according to a Ct value displayed by a fluorescent PCR amplification instrument. The addition and non-addition of the cfRNA settling agent can be judged to improve the extraction rate of the cfRNA by the qPCR verification of housekeeping genes, and the difference exists between the CT values.
The results are shown in the following table and in FIG. 1:
example 2
The free RNA precipitation aid provided by the embodiment consists of the following components: 5mg/ml dextran sulfate, 0.01mg/ml linear acrylamide and 0.1mg/ml acetate.
Aiming at the extraction of cfRNA of a pathogen-containing serum sample by a TRIzol reagent method, a one-step method qRT-PCR method is used for detecting the CT value of an internal reference 16srRNA gene, and the effect of adding and not adding a cfRNA settling agent is compared.
1. Nucleic acid extraction:
1) adding 0.5ml serum into 1ml TRIzol reagent, mixing, and standing at room temperature for 5-10 min.
2) 0.2ml of chloroform was added thereto, followed by vigorous shaking for 15 seconds and standing for 2 min.
3) Centrifuging at 4 deg.C for 12000g × 15min, and collecting supernatant.
4) Adding cfRNA precipitation aid with the addition of 100 mul, adding 0.5ml isopropanol, gently mixing the liquid in the tube, and standing at room temperature for 10 min. Meanwhile, only isopropanol was added without cfRNA precipitation aid in this step for comparison.
5) Centrifugation is carried out at 4 ℃ for 12000g × 10min, and the supernatant is discarded.
6) 1ml of 75% ethanol was added and the precipitate was washed gently. At 4 deg.C, 7500g × 5min, discard the supernatant.
7) Air drying, adding 30 μ l DEPC H2Dissolving O (promoting dissolution at 65 ℃ for 10-15 min).
8) Stored at-70 ℃ or used directly in the next experiment.
2. And (3) detection:
and (3) carrying out fluorescent quantitative PCR verification on the obtained cfRNA by using the reference gene 16srRNA, and judging a detection result according to the Ct value displayed by a fluorescent PCR amplification instrument. The addition and non-addition of the cfRNA settling agent can be judged to improve the extraction rate of the cfRNA by the qPCR verification of housekeeping genes, and the difference exists between the CT values.
The results are shown in the following table and fig. 2:
Claims (8)
1. a free RNA precipitation aid characterized by: each 1ml of the settling agent comprises the following components: 5-10mg dextran sulfate, 0.01-0.02mg linear acrylamide and 0.1-0.2mmol acetate.
2. A settling aid as claimed in claim 1, wherein: the acetate is ammonium acetate or potassium acetate.
3. A settling aid as claimed in claim 1, wherein: the settling agent is stored in a test tube at the temperature of-10-20 ℃.
4. Use of the free RNA precipitation aid of claim 1.
5. Use according to claim 4, characterized in that: the settling agent is used for extracting free RNA of a body fluid sample.
6. Use according to claim 5, characterized in that: the body fluid comprises plasma, serum, pleural fluid, ascites, cerebrospinal fluid or urine.
7. Use according to claim 4, characterized in that: the settling agent is used for the step of extracting a sample in a molecular biology downstream test.
8. Use according to claim 7, characterized in that: the molecular biological downstream assay comprises RT-PCR or qRT-PCR.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202011261598.1A CN112410402A (en) | 2020-11-12 | 2020-11-12 | Free RNA precipitation aid |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202011261598.1A CN112410402A (en) | 2020-11-12 | 2020-11-12 | Free RNA precipitation aid |
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101918594A (en) * | 2007-11-30 | 2010-12-15 | 俄亥俄州立大学研究基金会 | Micro-RNA expression profiling and targeting in peripheral blood in lung cancer |
| CN102690806A (en) * | 2012-05-02 | 2012-09-26 | 江苏大学 | Method for simply and rapidly extracting total RNA from switchgrass tissue |
| CN106399250A (en) * | 2015-07-31 | 2017-02-15 | 广州市锐博生物科技有限公司 | Method and kit for separating exosome |
| CN107405540A (en) * | 2014-10-24 | 2017-11-28 | 雅培分子公司 | The enrichment of small nucleic acids |
| WO2018090149A1 (en) * | 2016-11-21 | 2018-05-24 | Vivier Canada Inc. | Putrescine slow-release topical formulations |
| CN109679945A (en) * | 2018-07-26 | 2019-04-26 | 麦凯(上海)生物科技有限公司 | It is a kind of for improving the settling agent of dissociative DNA recovery rate in plasma sample |
-
2020
- 2020-11-12 CN CN202011261598.1A patent/CN112410402A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101918594A (en) * | 2007-11-30 | 2010-12-15 | 俄亥俄州立大学研究基金会 | Micro-RNA expression profiling and targeting in peripheral blood in lung cancer |
| CN102690806A (en) * | 2012-05-02 | 2012-09-26 | 江苏大学 | Method for simply and rapidly extracting total RNA from switchgrass tissue |
| CN107405540A (en) * | 2014-10-24 | 2017-11-28 | 雅培分子公司 | The enrichment of small nucleic acids |
| CN106399250A (en) * | 2015-07-31 | 2017-02-15 | 广州市锐博生物科技有限公司 | Method and kit for separating exosome |
| WO2018090149A1 (en) * | 2016-11-21 | 2018-05-24 | Vivier Canada Inc. | Putrescine slow-release topical formulations |
| CN109679945A (en) * | 2018-07-26 | 2019-04-26 | 麦凯(上海)生物科技有限公司 | It is a kind of for improving the settling agent of dissociative DNA recovery rate in plasma sample |
Non-Patent Citations (1)
| Title |
|---|
| ANTHONY F.JURITSCH等: "Rapid removal of dextran sulfate sodium from tissue RNA preparations for measurement of inflammation biomarkers", 《ANALYTICAL BIOCHEMISTRY》 * |
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Effective date of registration: 20210824 Address after: 314499 Room 101, 1f, Chuangye building, No. 3, No. 128 Shuanglian Road, Haining Economic Development Zone, Haining City, Jiaxing City, Zhejiang Province Applicant after: Haining Mackay medical laboratory Co.,Ltd. Address before: 201206 5th floor, 310 Jingao Road, Pudong New Area, Shanghai Applicant before: MAIKAI (SHANGHAI) BIOTECHNOLOGY Co.,Ltd. |
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