CN102676500A - Extraction and enrichment method of trace free mRNA (messenger Ribonucleic Acid) - Google Patents
Extraction and enrichment method of trace free mRNA (messenger Ribonucleic Acid) Download PDFInfo
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- CN102676500A CN102676500A CN2012100831896A CN201210083189A CN102676500A CN 102676500 A CN102676500 A CN 102676500A CN 2012100831896 A CN2012100831896 A CN 2012100831896A CN 201210083189 A CN201210083189 A CN 201210083189A CN 102676500 A CN102676500 A CN 102676500A
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Abstract
本发明涉及微量游离mRNA提取和富集方法。本发明是对含微量游离mRNA的液体离心取上清，加入到Trizol LS溶液中震荡，加入氯仿，剧烈震荡，离心，吸取水相层加入70％乙醇，混匀，转移到RNA pure link mini kit收集管中离心，使用wash buffer1和2洗涤滤网和滤网上的RNA，离心，干燥滤网和RNA，加无RNA酶水溶解RNA，离心，加DNA酶，终止DNA酶反应，离心，获得最终富集并纯化的RNA。本发明解决了血浆游离mRNA的检测存在检出率很低，标本量需求大，重复性差等缺陷。本发明从少量液体中抽提和富集微量(＜1ug)游离mRNA的方法，实时定量荧光PCR扩增证实相关目的基因的表达，并且显著提高了微量游离mRNA的检出率，操作方便，灵敏度高，特异性强，可重复性强。
The invention relates to a method for extracting and enriching trace free mRNA. The present invention is to centrifuge the liquid containing a small amount of free mRNA to take the supernatant, add it to the Trizol LS solution for shaking, add chloroform, shake vigorously, centrifuge, absorb the water phase layer, add 70% ethanol, mix well, and transfer it to the RNA pure link mini kit Centrifuge in the collection tube, use wash buffer 1 and 2 to wash the filter and the RNA on the filter, centrifuge, dry the filter and RNA, add RNase-free water to dissolve the RNA, centrifuge, add DNase, terminate the DNase reaction, centrifuge, and obtain the final Enriched and purified RNA. The invention solves the defects of low detection rate, large sample volume requirement, poor repeatability and the like in the detection of plasma free mRNA. The method of the present invention extracts and enriches a small amount of (<1ug) free mRNA from a small amount of liquid, real-time quantitative fluorescent PCR amplification confirms the expression of related target genes, and significantly improves the detection rate of a small amount of free mRNA, and is easy to operate and sensitive High, strong specificity, strong repeatability.
Description
Technical field
The present invention relates to a kind of medical science process for extracting, particularly micro free mRNA extracts and enriching method.
Background technology
In recent years discover; Free mRNA in the tumour patient blood plasma (cell-free mRNA) contains abundant tumor information;, can point out to have tumour cell in the blood or the indiscoverable micrometastasis of clinical imageology has taken place if in serum, detect like CK19mRNA; Colorectal carcinoma patient tumors tissue is significantly relevant with blood plasma hTERT-AT mRNA expression level, and all can reflect the degree of disease progression, and the prompting free mRNA can objectively respond tumor information; Patient with breast cancer's plasma free CyclinD1 is relevant with patient with breast cancer's prognosis and pharmacological agent curative effect with TS mRNA expression level.The discovery of plasma free nucleic acid provides new detection source with detection for the early diagnosis of tumour, monitoring in real time and real-time individuation pharmacological agent.
But because the existence of a large amount of RNA enzymes in the blood plasma, plasma free mRNA is very easy to degrade, and owing to the diluting effect of blood, therefore, the amount of every milliliter of plasma free mRNA considerably less (＜1ug).
Before the present invention, based on above two major causes, the detection of plasma free mRNA exists recall rate very low, and the specimen amount demand is big, problems such as poor repeatability.Thereby; The susceptibility that urgent need foundation possesses actual application value is high, specificity is high, micro free mRNA extracting, enrichment and the quantitative measurement technology of good reproducibility; Reach detection, to satisfy the needs of practical application to micro free mRNA contained in blood or other liquid.
Summary of the invention
The object of the invention just is to address the above problem, and develops a kind of micro free mRNA and extracts and enriching method.
Technical scheme of the present invention is:
Micro free mRNA extracts and enriching method, and its major technique step is:
(1) extract the liquid that contains micro free mRNA, two times centrifugal is obtained supernatant;
(2) get supernatant and join in the Trizol LS solution, the room temperature concussion;
(3) to wherein adding chloroform, concuss;
(4) centrifugal, draw aqueous phase layer to new pipe;
(5) add 70% ethanol, mixing; The liquid that will contain ethanol and RNA is transferred in the collection tube that has filter screen, and is centrifugal;
(6) RNA on washing filter screen and the filter screen;
(7) centrifugal, dry filter screen and RNA;
(8) add no RNA enzyme water dissolution RNA, centrifugal;
(9) add the DNA enzyme;
(10) stop the DNA enzyme reaction, centrifugal, obtain final enrichment and purified RNA, be template with it, reverse transcription is cDNA ,-20 ℃ of preservations.
Advantage of the present invention and effect be to invent a kind of from small amount of liquid (750ul) extracting and the gathering trace (＜1ug) method of free mRNA; The real-time quantitative fluorescence PCR amplification confirms the expression of relevant goal gene, and has significantly improved the recall rate of micro free mRNA.The present invention's tumor-related gene that can from a small amount of (750ul) plasma specimen, increase, can be from a small amount of (750ul) plasma specimen amplification chemotherapy genes involved, easy to operate, highly sensitive, high specificity, repeatable strong.Particularly:
1. broken through the method for from tumor tissues or tumour cell, extracting RNA traditionally, in a creative way successfully extracting from small amount of liquid, enrichment and detection by quantitative micro free mRNA;
2. the tumor specific expression gene can increase from a small amount of (750ul) plasma specimen;
3. can be from a small amount of (750ul) plasma specimen amplification related neoplasms affinity tag, like CEA, CA199, CA125 etc.;
4. chemotherapy genes involved (like BRCA1, ERCC1, hENT1, RRM1, TS, Topo1, APTX etc.) can increase from a small amount of (750ul) plasma specimen;
5. compare (the mRNA process for extracting reported of document is mostly based on RNA precipitation technology or enrichment with magnetic bead technology at present) with the free mRNA detection method of having reported at present, present method recall rate is high, and is highly sensitive; Repeatability is strong, high specificity, and cost is low; Easy and simple to handle, time saving and energy saving;
6. compare with the mRNA quantitative detecting method from the tumor tissues genes involved, present method has the great advantages of " in real time ", and this method is highly sensitive, and is easy and simple to handle, time saving and energy saving;
7. compare with the mRNA quantitative detecting method of genes involved in the circulating tumor cell, present method need not steps such as the separation, enrichment, cracking of loaded down with trivial details and complicated circulating tumor cell, and recall rate is high, highly sensitive, and repeatability is strong, and is easy and simple to handle, time saving and energy saving.
Description of drawings
The dissociate amplification synoptic diagram of BRCA1, ERCC1, hENT1, RRM1, TS, Topo1 and APTXmRNA of Fig. 1---part plasma specimen.
Fig. 2---BRCA1, ERCC1, hENT1, RRM1, TS, Topo1 and APTX mRNA are at the expression level and the recall rate contrast synoptic diagram of blood plasma.
Embodiment
Technological step of the present invention is following:
(1) all article are all through going the RNA enzyme to handle;
(2) 2ml blood is added in the no RNA enzyme pipe of 0.5ml 3.8% liquor sodii citratis;
(3) 3000g, 4 ℃ centrifugal 10 minutes, supernatant is transferred to new pipe;
(4) 12000g, once more 4 ℃ centrifugal 10 minutes, draw supernatant to new pipe;
(5) get the 750ul supernatant, add 2250ul Trizol LS, room temperature was fully shaken 10 minutes, to arrive the effect of abundant degraded nucleoprotein;
(6) every 3ml blood plasma-Trizol LS mixture adds chloroform 600ul, and room temperature was placed 5 minutes behind the concuss;
(7) 14000g, 4 ℃ centrifugal 15 minutes, draw aqueous phase layer to new pipe, be preliminary extractive RNA in this aqueous phase layer;
(8) add and isopyknic 70% ethanol of aqueous phase layer, and mixing;
The liquid that (9) will contain ethanol and RNA transfer to have filter screen collection tube 1. in (Ambion product; Article No. 12183-018A), 700ul once, 12000g; Centrifugal 1.5 minutes of room temperature; Discard centrifugal gained liquid, then repeatedly with remaining ethanol and RNA mixing liquid transfer to have filter screen collection tube 1. in, finish until all mixing liquids are all centrifugal.The purpose of this step be all RNA enrichments to filter screen;
(10) discard collection tube 1., keep filter screen, and 2. filter screen is put into another clean collection tube, add 700ul washing composition wash buffer 1 (Ambion product, article No. 12183-018A), 12000g, centrifugal 1.5 minutes of room temperature is to remove residual ethanol.
(11) discard collection tube and 2. reach liquid in pipe, keep filter screen, and 3. filter screen is put into another new collection tube; Add 500ul washing composition wash buffer2 (Ambion product, article No. 12183-018A), 12000g; Centrifugal 1.5 minutes of room temperature is to remove residual washing composition wash buffer1;
(12) repeating step (12);
(13) discard 3. and wherein residual liquid of collection tube, and 4. filter screen is put into another clean new collection tube, 14000g, centrifugal 2.5 minutes of room temperature reaches the purpose of RNA on dry filter screen and the filter screen;
(14) discard collection tube and 4. reach liquid in pipe, keep filter screen, with filter screen insert another clean new collection tube 5. in, on filter screen, adding 33ul does not have RNA enzyme water, incubated at room 1 minute reaches the purpose of RNA on the abundant dissolving filter screen;
(15) 14000g, centrifugal 3 minutes of room temperature, this moment, the RNA on the filter screen was all centrifugal to 5. bottom of collection tube, obtained the RNA of extracting and enrichment;
(16) 5. middle 10 * DNA enzyme buffer liquid 6ul and the 1.5ul DNA enzyme of adding of collection tube, mixing was hatched 30 minutes for 37 ℃, reached the purpose of removing the potential minim DNA, fully purifying RNA;
(17) add the agent of 6ul DNA enzyme-deactivating, after room temperature is placed, 11000g, 4 ℃ centrifugal 1 minute, with deactivation DNA enzyme;
(18) the absorption supernatant reaches and is final extraction, enrichment and purified RNA, is template with it, is cDNA by the reverse transcription of InvitrogenM-MLV reverse transcription test kit description operation ,-20 ℃ of preservations.
Adopt Taqman-MGB probe technique real-time fluorescence quantitative PCR to detect the expression (is confidential reference items with β-actin) of blood plasma micro free BRCA1, ERCC1, hENT1, RRM1, TS, Topo1 and APTX mRNA; Used probe and primer are ABI product (primer is striden the intron design, in amplification, has avoided the DNA contamination of heavy), and the amplification of genes involved has confirmed successful extraction and the enrichment (Fig. 1) of blood plasma micro free mRNA.X-coordinate is the PCR cycle number among Fig. 1, and ordinate zou is a fluorescence intensity.All goal gene are all effectively increased and are detected; Each detection by quantitative is equipped with the positive and negative control, and negative control is not seen amplification, has got rid of the sample DNA contamination of heavy.
What be worth to stress is; The present invention has only used a small amount of (750ul) plasma specimen just to realize effective amplification of micro-goal gene and successfully detect; And having significantly improved the recall rate of blood plasma micro free mRNA---our method makes the recall rate of blood plasma TS mRNA bring up to 92%, is higher than the recall rate 55% that present additive method has been reported far away; We have also successfully detected other 6 biological markers (BRCA1, ERCC1, hENT1, RRM1, Topo1 and APTX) with important value from blood plasma; And these genes do not have bibliographical information from blood plasma, successfully to detect because its expression level is low so far as yet.Expression level and the recall rate contrast of these genes in blood plasma is as shown in Figure 2.Our this technology is that solid foundation has been established in further laboratory of plasma free mRNA and clinical application.
According to the principle of the invention and experimental data, the present invention also can be used for the detection by quantitative of other sick kinds and other micro frees mRNA.The scope that the present invention asks for protection is not limited only to the description of this embodiment.
Claims (5)
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115851943A (en) * | 2022-11-17 | 2023-03-28 | 杭州艾迪康医学检验中心有限公司 | Reagent and method for detecting TOP1 gene expression level |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1952176A (en) * | 2006-09-27 | 2007-04-25 | 南京大学医学院附属鼓楼医院 | Process for extracting free mRNA from pleural fluid and ascitic fluid |
| WO2009149026A9 (en) * | 2008-06-01 | 2010-04-01 | Tufts Medical Center, Inc. | Genomic approaches to fetal treatment and diagnosis |
| WO2011154940A1 (en) * | 2010-06-07 | 2011-12-15 | Osnat Ashur-Fabian | Methods and kits for diagnosing conditions related to hypoxia |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1952176A (en) * | 2006-09-27 | 2007-04-25 | 南京大学医学院附属鼓楼医院 | Process for extracting free mRNA from pleural fluid and ascitic fluid |
| WO2009149026A9 (en) * | 2008-06-01 | 2010-04-01 | Tufts Medical Center, Inc. | Genomic approaches to fetal treatment and diagnosis |
| WO2011154940A1 (en) * | 2010-06-07 | 2011-12-15 | Osnat Ashur-Fabian | Methods and kits for diagnosing conditions related to hypoxia |
Non-Patent Citations (1)
| Title |
|---|
| 黄诗韵: "精浆游离mRNA一般特征及附睾液游离mRNA功能的初步研究", 《中国博士学位论文全文数据库医药卫生科技辑》, 15 November 2010 (2010-11-15), pages 067 - 17 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115851943A (en) * | 2022-11-17 | 2023-03-28 | 杭州艾迪康医学检验中心有限公司 | Reagent and method for detecting TOP1 gene expression level |
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Application publication date: 20120919 |