CN102127600A - Method for detecting piwi-interacting ribonucleic acid (piRNA) in gastric juice - Google Patents

Abstract

Translated fromChinese

本发明公开了一种胃液中piRNA的检测方法，包括胃液抽取和中和、RNA释放、氯仿提取、异丙醇沉淀、乙醇洗涤、RNA逆转录、PCR检测等步骤，该检测方法使用氢氧化钠溶液，对胃液中的粘稠成分进行液化使粘蛋白和多糖悬于溶液中，并中和胃液；加入胰蛋白酶进行消化，可以使粘蛋白等蛋白质水解，同时使胃液中的细胞成分变得松散，便于离心分离；加入Trizol溶液反复吹打，RNA充分释放并与多糖和粘蛋白等杂质分离，解决了胃液中酸度、多糖和粘蛋白等对RNA提取的干扰问题，因此本发明可以去除胃液中多糖和蛋白质等杂质干扰，对胃液中piRNA的有效提取，并对提取的piRNA进行荧光定量PCR检测。

The invention discloses a method for detecting piRNA in gastric juice, which includes the steps of gastric juice extraction and neutralization, RNA release, chloroform extraction, isopropanol precipitation, ethanol washing, RNA reverse transcription, PCR detection and the like. The detection method uses sodium hydroxide Solution, to liquefy the viscous components in gastric juice, suspend mucin and polysaccharide in the solution, and neutralize gastric juice; add trypsin for digestion, it can hydrolyze mucin and other proteins, and at the same time make the cellular components in gastric juice loose , easy to centrifuge; adding Trizol solution repeatedly blowing, RNA is fully released and separated from impurities such as polysaccharides and mucins, which solves the interference problem of acidity, polysaccharides and mucins in gastric juice on RNA extraction, so the present invention can remove polysaccharides in gastric juice Interference with impurities such as proteins, effective extraction of piRNA in gastric juice, and fluorescent quantitative PCR detection of the extracted piRNA.

Description

Translated fromChinese

一种胃液中piRNA的检测方法A method for detecting piRNA in gastric juice

技术领域technical field

本发明涉及piRNA的检测方法，具体涉及一种胃液中piRNA的检测方法。The invention relates to a method for detecting piRNA, in particular to a method for detecting piRNA in gastric juice.

背景技术Background technique

生物体中的RNA可以分为两大类：编码RNA和非编码RNA，人们发现非编码RNA（non-coding RNA, ncRNA）参与组成了细胞中高度复杂的RNA调控网络。它们在调节细胞增殖、发育时序、细胞免疫和疾病发生等过程中发挥着重要功能。在这些非编码RNA中，Piwi相互作用RNA（Piwi-interacting RNA, piRNA）具有调节生殖细胞的形成、性别决定、参与干细胞定向分化、维持转座子抑制状态和防止转座子爆发等多种功能。RNA in organisms can be divided into two categories: coding RNA and non-coding RNA. It has been found that non-coding RNA (non-coding RNA, ncRNA) participates in the highly complex RNA regulatory network in cells. They play important functions in the regulation of cell proliferation, developmental timing, cellular immunity, and disease occurrence. Among these non-coding RNAs, Piwi-interacting RNA (piRNA) has multiple functions such as regulating the formation of germ cells, sex determination, participating in the directed differentiation of stem cells, maintaining the repressed state of transposons, and preventing transposon outbreaks. .

piRNA是一类长度为25~31个核苷酸、不编码蛋白质的单链小RNA分子。它们的生成不依赖于Dicer酶的加工，而通过与阿格蛋白（argonaute protein）亚家族中的Piwi蛋白相互结合来发挥调控mRNA的稳定性和染色质形成等重要生物学功能。piRNA is a class of single-stranded small RNA molecules with a length of 25-31 nucleotides and no protein coding. Their production does not depend on the processing of Dicer enzymes, but by combining with Piwi proteins in the argonaute protein subfamily, they play important biological functions such as regulating mRNA stability and chromatin formation.

对于piRNA的分析需要小RNA提取和检测，小RNA提取如中国专利申请号为200810120866.0，名称为“一种从鼠脑组织中获得小分子RNA的方法”，就公开了包括组织研磨，离心去不溶物，氯仿抽提：氯仿与Ezol 0.25：1，振荡15秒，静置5分钟后离心，异丙醇沉淀：-15～-20℃下沉淀12小时，离心，用75%乙醇洗涤沉淀，离心后用DEPC-双蒸水溶解，得到小RNA提取液。小RNA检测包括早期的Northern印迹法，但费时费力，需在大量的总RNA标本；聚合酶链式反应（polymerase chain reaction, PCR）是一种准确、定量检测方法，但由于小RNA长度很短，以至于不能通过经典PCR法直接扩增，需要在小RNA尾端连接多个腺嘌呤来延长长度或使用较长探针或几个连接在一起的探针组，才可检测，如中国专利申请号为200910084474.8，名称为“用于小RNA检测的探针组以及用此探针组检测小RNA的方法”的发明专利申请，就公开了用至少具有一个茎环结构的探针，多个这样探针连接起来组成探针组进行小RNA检测。The analysis of piRNA requires the extraction and detection of small RNA, such as Chinese patent application number 200810120866.0, titled "A Method for Obtaining Small RNA from Rat Brain Tissue", which includes tissue grinding, centrifugation to remove insoluble Chloroform extraction: Chloroform and Ezol 0.25:1, shaking for 15 seconds, centrifuging after standing for 5 minutes, precipitation with isopropanol: precipitation at -15～-20°C for 12 hours, centrifugation, washing with 75% ethanol, centrifugation Then dissolve with DEPC-double distilled water to obtain small RNA extract. Small RNA detection includes the early Northern blot method, but it is time-consuming and labor-intensive, requiring a large number of total RNA samples; polymerase chain reaction (polymerase chain reaction, PCR) is an accurate and quantitative detection method, but due to the short length of small RNA , so that it cannot be directly amplified by the classic PCR method, it is necessary to connect multiple adenines at the end of the small RNA to extend the length or use a longer probe or several probe groups connected together to detect, such as the Chinese patent The application number is 200910084474.8, and the invention patent application named "probe set for small RNA detection and method for detecting small RNA with this probe set" discloses the use of probes with at least one stem-loop structure, multiple In this way, the probes are connected to form a probe set for small RNA detection.

胃液的一个重要特点是含有大量的壁细胞分泌的盐酸，其pH值约为0.9-1.5，具有强酸性。胃液成分也相当复杂，除了含有食物残渣外，还含有胃蛋白酶、核酸酶、粘液、内因子等有机物，其中核酸酶较为稳定，有可能分解RNA，这些因素均影响到RNA的提取和检测。另外胃液中存在大量的粘多糖和粘蛋白等物质，在运用常规方法提取RNA的过程中，由于多糖的理化性质和RNA非常相似，多糖和RNA可以同时沉淀下来，产生富集多糖的透明胶状沉淀，不利于后续的cDNA合成和PCR检测。An important feature of gastric juice is that it contains a large amount of hydrochloric acid secreted by parietal cells, and its pH value is about 0.9-1.5, which is strongly acidic. The composition of gastric juice is also quite complex. In addition to food residues, it also contains organic substances such as pepsin, nuclease, mucus, and intrinsic factor. Among them, nuclease is relatively stable and may decompose RNA. These factors affect the extraction and detection of RNA. In addition, there are a lot of substances such as mucopolysaccharides and mucins in the gastric juice. During the process of extracting RNA by conventional methods, because the physical and chemical properties of polysaccharides are very similar to RNA, polysaccharides and RNA can be precipitated at the same time, resulting in a transparent gel rich in polysaccharides. Precipitation is not conducive to subsequent cDNA synthesis and PCR detection.

胃液中piRNA的含量很低，因此对胃液中piRNA的提取和检测难度很大，目前还没有具体提取和胃液中piRNA的检测方法。The content of piRNA in gastric juice is very low, so it is very difficult to extract and detect piRNA in gastric juice, and there is no specific extraction and detection method for piRNA in gastric juice.

发明内容Contents of the invention

本发明所要解决的技术问题是提供一种胃液中piRNA的检测方法，该检测方法能去除胃液中多糖和蛋白质等杂质干扰，对胃液中piRNA的有效提取，并对提取的piRNA进行荧光定量RT-PCR检测。The technical problem to be solved by the present invention is to provide a detection method for piRNA in gastric juice, which can remove the interference of impurities such as polysaccharides and proteins in gastric juice, effectively extract piRNA in gastric juice, and perform fluorescence quantitative RT- PCR detection.

本发明解决上述技术问题所采用的技术方案为：一种胃液中piRNA的检测方法，包括下述步骤：The technical solution adopted by the present invention to solve the above-mentioned technical problems is: a method for detecting piRNA in gastric juice, comprising the following steps:

a、胃液抽取和中和：用胃管抽取空腹胃液5ml，放入离心管中，置于冰上，然后加入质量百分浓度为20%的氢氧化钠溶液0.5ml，置摇床中常温振摇20分钟，可以使胃液中粘液液化，用质量百分浓度为20%的氢氧化钠溶液调整pH值至7.0±0.5，再加入质量百分浓度为1%的胰蛋白酶溶液20μl，漩涡混匀，置37℃水浴箱中消化15分钟，可以让胃液中的蛋白质水解并使胃液均质化，4℃下，1000rpm离心20分钟；a. Extraction and neutralization of gastric juice: extract 5ml of gastric juice on an empty stomach with a gastric tube, put it into a centrifuge tube, place it on ice, then add 0.5ml of sodium hydroxide solution with a concentration of 20% by mass, and shake it in a shaker at room temperature. Shake for 20 minutes to liquefy the mucus in gastric juice, adjust the pH value to 7.0±0.5 with 20% sodium hydroxide solution by mass percent, then add 20 μl of trypsin solution with 1% mass percent concentration, and vortex to mix , placed in a 37°C water bath for 15 minutes to hydrolyze the protein in the gastric juice and homogenize the gastric juice, centrifuge at 1000rpm for 20 minutes at 4°C;

b、RNA释放：取步骤a的上清液200μl置于消毒离心管中，加入50μl灭菌的焦碳酸乙二酯（Diethylpyrocarbonate，DEPC）处理过的双蒸水，混匀，再加入Trizol溶液1ml，反复吹打20次，室温下静置10分钟，可以让RNA充分释放到溶液中，4℃下，12000rpm离心15分钟，丢弃沉淀，保留上清液，以除去残余的多糖和粘蛋白；b. RNA release: Take 200 μl of the supernatant from step a and place it in a sterile centrifuge tube, add 50 μl of sterilized double distilled water treated with Diethylpyrocarbonate (DEPC), mix well, and then add 1ml of Trizol solution , repeatedly pipetting 20 times, standing at room temperature for 10 minutes, can fully release the RNA into the solution, centrifuge at 12000rpm for 15 minutes at 4°C, discard the precipitate, and keep the supernatant to remove residual polysaccharides and mucins;

c、氯仿提取：将步骤b的上清液移入另一消毒离心管中，加入0.2ml氯仿，漩涡振荡15秒，室温下静置5分钟，4℃下，12000rpm离心15分钟，吸取上层水相移入DEPC处理过的灭菌离心管中；c. Chloroform extraction: transfer the supernatant from step b into another sterilized centrifuge tube, add 0.2ml chloroform, vortex for 15 seconds, let stand at room temperature for 5 minutes, centrifuge at 12000rpm for 15 minutes at 4°C, and absorb the upper aqueous phase Transfer to DEPC-treated sterilized centrifuge tubes;

d、异丙醇沉淀：在步骤c 的DEPC处理过的灭菌离心管中加入与所述上层水相等体积的异丙醇，混匀，室温下静置20分钟，4℃下，12000rpm离心15分钟，弃上清得沉淀；d. Isopropanol precipitation: add isopropanol equal to the volume of the upper layer of water to the DEPC-treated sterilized centrifuge tube in step c, mix well, let stand at room temperature for 20 minutes, and centrifuge at 12,000 rpm for 15 minutes at 4°C. minutes, discard the supernatant to obtain the precipitate;

e、乙醇洗涤：在步骤d的沉淀中，加入质量百分浓度为75%的乙醇1ml，颠倒混匀20次洗涤，4℃下，12000rpm离心3分钟，弃上清，再用同浓度和同量的乙醇，同样重复洗涤一次，4℃下，12000rpm离心3分钟，弃上清，室温下干燥10分钟，加15μl双蒸水溶解，得到RNA提取液，-80℃保存备用；e. Washing with ethanol: Add 1ml of ethanol with a mass percentage concentration of 75% to the precipitation in step d, invert and mix for 20 washes, centrifuge at 12000rpm for 3 minutes at 4°C, discard the supernatant, and use the same concentration and the same Wash again with a large amount of ethanol, centrifuge at 12,000 rpm for 3 minutes at 4°C, discard the supernatant, dry at room temperature for 10 minutes, add 15 μl of double-distilled water to dissolve, and obtain RNA extract, which is stored at -80°C for later use;

f、RNA逆转录：用miScript 逆转录（reverse transcription, RT）试剂盒（购于德国Qiagen公司）对步骤e的RNA提取液进行逆转录反应，得到cDNA溶液，置-20℃保存备用，具体操作依该试剂盒说明书进行：在0.5ml离心管中加入10μl RNA提取液、5μl DEPC双蒸水、1μl 逆转录混合试剂和4μl RT 缓冲液；然后在37℃保温1小时，再在95℃保温5分钟，加入80μl DEPC双蒸水，就得到cDNA溶液；f. RNA reverse transcription: use miScript reverse transcription (reverse transcription, RT) kit (purchased from Qiagen, Germany) to carry out reverse transcription reaction on the RNA extraction solution in step e to obtain a cDNA solution, and store it at -20°C for later use. The specific operation Follow the instructions of the kit: add 10 μl RNA extraction solution, 5 μl DEPC double distilled water, 1 μl reverse transcription mixed reagent and 4 μl RT buffer to a 0.5ml centrifuge tube; Minutes, add 80 μl DEPC double distilled water, just get cDNA solution;

g、PCR检测：用miScript SYBR Green PCR检测试剂盒（购于德国Qiagen公司）对步骤f的cDNA溶液进行PCR扩增反应，扩增的上游引物为piRNA特异性扩增上游引物或小RNA U6特异性扩增上游引物（购于德国Qiagen公司），扩增的下游引物为小RNA通用下游引物（购于德国Qiagen公司），再用荧光定量PCR仪（购于美国Stratagene公司）检测，具体操作依检测试剂盒和荧光定量PCR仪说明书进行：在薄壁透明荧光定量PCR反应管中分别加入1μl piRNA特异性扩增上游引物或小RNA U6特异性扩增上游引物和1μl PCR检测的小RNA通用下游引物，6μl cDNA溶液，10μl SYBR Green PCR反应缓冲液，2μl DEPC双蒸水；PCR反应条件：95℃预变性15分钟；然后94℃变性15秒，60℃退火30秒，70℃延伸30秒，共40个循环；解链曲线分析：95 ℃ 1分钟，55℃ 30秒；然后缓慢升温至95℃，升温速率为0.2℃/秒。最后得到扩增曲线图和解链曲线图，从扩增曲线的Ct值可以得到各piRNA的表达水平，从解链曲线中可以了解到其曲线为较窄的单一峰，可以看出每种piRNA均被特异性扩增，没有引物二聚体和杂带的干扰。g. PCR detection: Use miScript SYBR Green PCR detection kit (purchased from Qiagen, Germany) to perform PCR amplification reaction on the cDNA solution in step f, and the amplified upstream primers are piRNA-specific amplification upstream primers or small RNA U6-specific (purchased from Qiagen, Germany), and the amplified downstream primers are general downstream primers for small RNA (purchased from Qiagen, Germany), and then detected by a fluorescent quantitative PCR instrument (purchased from Stratagene, USA). Detection kit and fluorescent quantitative PCR instrument instructions: add 1 μl piRNA specific amplification upstream primer or small RNA U6 specific amplification upstream primer and 1 μl PCR detection small RNA general downstream primer to the thin-walled transparent fluorescent quantitative PCR reaction tube Primers, 6 μl cDNA solution, 10 μl SYBR Green PCR reaction buffer, 2 μl DEPC double distilled water; PCR reaction conditions: pre-denaturation at 95°C for 15 minutes; then denaturation at 94°C for 15 seconds, annealing at 60°C for 30 seconds, extension at 70°C for 30 seconds, A total of 40 cycles; melting curve analysis: 95°C for 1 minute, 55°C for 30 seconds; then slowly increase the temperature to 95°C with a heating rate of 0.2°C/s. Finally, the amplification curve and the melting curve are obtained. From the Ct value of the amplification curve, the expression level of each piRNA can be obtained. From the melting curve, it can be understood that its curve is a narrow single peak. It can be seen that each piRNA has It is specifically amplified without the interference of primer dimers and heterogeneous bands.

所述piRNA特异性扩增上游引物为has-piR-651扩增上游引物（购于上海吉玛制药技术有限公司），具体序列为5’AGAGAGGGGC CCGTGCCTTG 3’。The piRNA-specific amplification upstream primer is the has-piR-651 amplification upstream primer (purchased from Shanghai Gemma Pharmaceutical Technology Co., Ltd.), and the specific sequence is 5' AGAGAGGGGC CCGTGCCTTG 3'.

所述piRNA特异性扩增上游引物为has-piR-823扩增上游引物（购于上海吉玛制药技术有限公司），具体序列为5’ AGCGTTGGTG GTATAGTGGT 3’。The piRNA-specific amplification upstream primer is the has-piR-823 amplification upstream primer (purchased from Shanghai Gemma Pharmaceutical Technology Co., Ltd.), and the specific sequence is 5' AGCGTTGGTG GTATAGTGGT 3'.

与现有技术相比，本发明的优点在于一种胃液中piRNA的检测方法，通过胃液抽取和中和、释放RNA、氯仿提取、异丙醇沉淀、乙醇洗涤、RNA逆转录和PCR检测步骤，使用氢氧化钠溶液，对胃液中的粘稠成分进行液化使粘蛋白和多糖悬于溶液中，并中和胃液；加入胰蛋白酶进行消化，可以使粘蛋白等蛋白质水解，同时使胃液中的细胞成分变得松散，便于离心分离；加入Trizol溶液反复吹打，RNA充分释放并与多糖和粘蛋白等杂质分离，通过离心使多糖等杂质沉于管底达到去除目的；这些操作有效地解决了胃液中酸度、多糖和粘蛋白等对RNA提取的干扰，而采用miScript SYBR Green PCR检测试剂盒进行PCR检测也解决了胃液中piRNA含量较少的检测问题；因此本发明可以去除胃液中多糖和蛋白质等杂质干扰，对胃液中piRNA的有效提取，并对提取的piRNA无需延长就可进行荧光定量PCR检测。Compared with the prior art, the present invention has the advantage of a method for detecting piRNA in gastric juice, through gastric juice extraction and neutralization, release of RNA, chloroform extraction, isopropanol precipitation, ethanol washing, RNA reverse transcription and PCR detection steps, Use sodium hydroxide solution to liquefy the viscous components in gastric juice, suspend mucin and polysaccharides in the solution, and neutralize gastric juice; add trypsin for digestion, which can hydrolyze mucin and other proteins, and at the same time make cells in gastric juice The components become loose and easy to be separated by centrifugation; adding Trizol solution and pipetting repeatedly, the RNA is fully released and separated from impurities such as polysaccharides and mucins, and the impurities such as polysaccharides sink to the bottom of the tube through centrifugation to achieve the purpose of removal; these operations effectively solve the problem of gastric juice Acidity, polysaccharides and mucin, etc. interfere with RNA extraction, and using miScript SYBR Green PCR detection kit for PCR detection also solves the detection problem of less piRNA content in gastric juice; therefore, the present invention can remove impurities such as polysaccharides and proteins in gastric juice Interference, effective extraction of piRNA in gastric juice, and fluorescence quantitative PCR detection can be performed on the extracted piRNA without extension.

附图说明Description of drawings

图1为实施例1的两个胃液标本中的piR-651扩增曲线图；Fig. 1 is the piR-651 amplification curve in two gastric juice samples ofembodiment 1;

图2为实施例1的两个胃液标本中的piR-651解链曲线图；Fig. 2 is the melting curve diagram of piR-651 in two gastric juice specimens ofembodiment 1;

图3为实施例2的两个胃液标本中的p iR-823扩增曲线图；Fig. 3 is the piR-823 amplification curve figure in two gastric juice specimens ofembodiment 2;

图4为实施例2的两个胃液标本中的p iR-823解链曲线图；Fig. 4 is the melting curve figure of piR-823 in two gastric juice specimens ofembodiment 2;

图5为实施例3的两个胃液标本中的小RNA U6扩增曲线图；Fig. 5 is the small RNA U6 amplification curve figure in two gastric juice specimens of embodiment 3;

图6为实施例3的两个胃液标本中的小RNA U6解链曲线图。Fig. 6 is the melting curve diagram of small RNA U6 in two gastric juice specimens of embodiment 3.

具体实施方式Detailed ways

实施例1Example 1

一种胃液中piRNA的检测方法，依次包括以下主要步骤：A method for detecting piRNA in gastric juice, comprising the following main steps in sequence:

a、胃液抽取和中和：用胃管抽取二个空腹胃液标本(不同部位)各5ml，放入离心管中，置于冰上，然后加入质量百分浓度为20%的氢氧化钠溶液0.5ml，置摇床中常温振摇20分钟，可以使胃液中粘液液化，用质量百分浓度为20%的氢氧化钠溶液调整pH值至7.0±0.5，再加入质量百分浓度为1%的胰蛋白酶（市售）溶液20μl，漩涡混匀，置37℃水浴箱中消化15分钟，使胃液中的蛋白质水解并使胃液均质化，4℃下，1000rpm离心20分钟；a. Gastric juice extraction and neutralization: extract two fasting gastric juice samples (different parts) with a gastric tube, 5ml each, put them into a centrifuge tube, put them on ice, and then add 0.5% sodium hydroxide solution with a mass percentage concentration of 20%. ml, placed in a shaker at room temperature and shaken for 20 minutes to liquefy the mucus in gastric juice, adjust the pH value to 7.0±0.5 with 20% sodium hydroxide solution by mass percentage, and then add 1% Trypsin (commercially available) solution 20μl, vortex to mix, place in a 37℃ water bath for 15 minutes to digest, hydrolyze the protein in the gastric juice and homogenize the gastric juice, centrifuge at 1000rpm for 20 minutes at 4℃;

b、RNA释放：取步骤a的上清液200μl置于消毒离心管中，加入50μl灭菌的DEPC（市售）处理过的双蒸水，混匀，再加入Trizol溶液（市售）1ml，反复吹打20次，室温下静置10分钟，使RNA充分释放到溶液中，4℃下，12000rpm离心15分钟，丢弃沉淀，保留上清液，以除去残余的多糖和粘蛋白；b. RNA release: Take 200 μl of the supernatant from step a and place it in a sterile centrifuge tube, add 50 μl of sterilized DEPC (commercially available) treated double distilled water, mix well, then add 1ml of Trizol solution (commercially available), Pipette repeatedly 20 times, let stand at room temperature for 10 minutes to fully release the RNA into the solution, centrifuge at 12,000 rpm for 15 minutes at 4°C, discard the precipitate, and keep the supernatant to remove residual polysaccharides and mucins;

c、氯仿提取：将步骤b的上清液移入另一消毒离心管中，加入0.2ml氯仿，漩涡振荡15秒，室温下静置5分钟；4℃下，12000rpm离心15分钟；吸取上层水相移入DEPC处理过的灭菌离心管中；c. Chloroform extraction: transfer the supernatant from step b into another sterilized centrifuge tube, add 0.2ml chloroform, vortex for 15 seconds, and let stand at room temperature for 5 minutes; centrifuge at 12000rpm for 15 minutes at 4°C; absorb the upper aqueous phase Transfer to DEPC-treated sterilized centrifuge tubes;

d、异丙醇沉淀：在上述DEPC处理过的灭菌离心管中加入与上层水相等体积的异丙醇，混匀，室温下静置20分钟；4℃下，12000rpm离心15分钟，弃上清得沉淀；d. Isopropanol precipitation: Add isopropanol equal in volume to the upper layer of water in the above-mentioned DEPC-treated sterilized centrifuge tube, mix well, and let stand at room temperature for 20 minutes; centrifuge at 12,000 rpm for 15 minutes at 4°C, discard the upper layer clear sediment;

e、乙醇洗涤：在步骤d的沉淀中，加入质量百分浓度为75%的乙醇1ml，颠倒混匀20次洗涤；4℃下，12000rpm离心3分钟；弃上清，再用质量百分浓度为75%的乙醇1ml同样重复洗涤一次，4℃下，12000rpm离心3分钟，弃上清；室温下干燥10分钟，加15μl双蒸水溶解，得到RNA提取液，-80℃保存备用；e. Washing with ethanol: Add 1ml of ethanol with a mass percentage concentration of 75% to the precipitation in step d, invert and mix for 20 washes; at 4°C, centrifuge at 12000rpm for 3 minutes; discard the supernatant, and then use the mass percentage concentration Wash again with 1ml of 75% ethanol, centrifuge at 12,000 rpm for 3 minutes at 4°C, discard the supernatant; dry at room temperature for 10 minutes, add 15 μl of double distilled water to dissolve, and obtain RNA extract, store at -80°C for later use;

f、RNA逆转录：用miScript 逆转录试剂盒对步骤e的RNA提取液进行逆转录反应，具体操作依该试剂盒说明书进行：在0.5ml离心管中加入10μl RNA提取液、5μl DEPC双蒸水、1μl 逆转录混合试剂和4μl RT 缓冲液；然后在37℃保温1小时，再在95℃保温5分钟，加入80μl DEPC双蒸水，得到cDNA溶液，置-20℃保存备用；f. RNA reverse transcription: use the miScript reverse transcription kit to carry out reverse transcription reaction on the RNA extraction solution in step e, and the specific operation is carried out according to the kit instructions: add 10 μl RNA extraction solution and 5 μl DEPC double-distilled water to a 0.5ml centrifuge tube , 1 μl reverse transcription mixed reagent and 4 μl RT buffer; then incubate at 37°C for 1 hour, then incubate at 95°C for 5 minutes, add 80 μl DEPC double distilled water to obtain cDNA solution, store at -20°C for later use;

g、PCR检测：用miScript SYBR Green PCR检测试剂盒对步骤f的cDNA溶液进行PCR扩增反应，扩增引物为piR-651扩增上游引物和PCR检测的小RNA通用下游引物，再用荧光定量PCR仪检测，具体操作依检测试剂盒和荧光定量PCR仪说明书进行：在薄壁透明荧光定量PCR反应管中分别加入1μl piR-651扩增上游引物和1μl PCR检测的小RNA通用下游引物，6 μl cDNA溶液，10μl SYBR Green PCR反应缓冲液，2μl DEPC双蒸水；PCR反应条件：95℃预变性15分钟；然后94℃变性15秒，60℃退火30秒，70℃延伸30秒，共40个循环；解链曲线分析：95 ℃ 1分钟，55℃ 30秒；然后缓慢升温至95℃，升温速率为0.2℃/秒。最后得到如附图1所示的piR-651扩增曲线和如附图2所示的piR-651解链曲线，从扩增曲线可以得到所检测的二个胃液标本的Ct值分别为25.04和26.28，从解链曲线中可以了解到该曲线为较窄的单一峰，可以看出每个胃液标本的piR-651均被特异性扩增，没有引物二聚体和杂带的干扰，PCR检测时无需在小RNA尾端连接多个腺嘌呤。g. PCR detection: use miScript SYBR Green PCR detection kit to carry out PCR amplification reaction on the cDNA solution in step f, the amplification primers are piR-651 amplification upstream primers and small RNA general downstream primers for PCR detection, and then use fluorescence quantification PCR instrument detection, the specific operation is carried out according to the detection kit and fluorescent quantitative PCR instrument manual: add 1 μl piR-651 amplification upstream primer and 1 μl small RNA general downstream primer for PCR detection respectively into the thin-walled transparent fluorescent quantitative PCR reaction tube, 6 μl cDNA solution, 10 μl SYBR Green PCR reaction buffer, 2 μl DEPC double distilled water; PCR reaction conditions: pre-denaturation at 95°C for 15 minutes; then denaturation at 94°C for 15 seconds, annealing at 60°C for 30 seconds, extension at 70°C for 30 seconds, a total of 40 cycle; melting curve analysis: 95°C for 1 minute, 55°C for 30 seconds; then slowly increase the temperature to 95°C with a heating rate of 0.2°C/sec. Obtain at last the piR-651 amplification curve as shown in accompanying drawing 1 and the piR-651 melting curve as shown in accompanying drawing 2, can obtain the Ct value of two gastric juice samples that detect from amplification curve to be 25.04 and 25.04 respectively. 26.28. From the melting curve, it can be seen that the curve is a narrow single peak. It can be seen that the piR-651 of each gastric juice sample is amplified specifically, without the interference of primer dimers and heterogeneous bands. PCR detection There is no need to attach multiple adenines to the tail of the small RNA.

实施例2Example 2

与实施例1方法基本相同，所不同的只是在步骤g 中，扩增的上游引物由piR-823特异性扩增上游引物代替piR-651，检测得到胃液中piR-823表达水平的扩增曲线（附图3）和解链曲线（附图4），从扩增曲线可以得到所检测的二个胃液标本的Ct值分别为22.27和22.91；从解链曲线中可以了解到该曲线为较窄的单一峰，可以看出每个胃液标本的piR-823均被特异性扩增，没有引物二聚体和杂带的干扰。The method is basically the same as in Example 1, except that in step g, the amplified upstream primer is replaced by the piR-823 specific amplification upstream primer for piR-651, and the amplification curve of the expression level of piR-823 in the gastric juice is detected (Figure 3) and melting curve (Figure 4), from the amplification curve, the Ct values of the two gastric juice samples detected were 22.27 and 22.91 respectively; from the melting curve, it can be known that the curve is narrow Single peak, it can be seen that piR-823 of each gastric juice sample is amplified specifically, without the interference of primer dimers and heterogeneous bands.

实施例3Example 3

与实施例1方法基本相同，所不同的只是在步骤g 中，扩增的上游引物由小RNA U6特异性扩增上游引物代替piR-651，检测得到胃液中小RNA U6表达水平的扩增曲线（附图5）和解链曲线（附图6），从扩增曲线可以得到所检测的二个胃液标本的Ct值分别为29.60和32.04；从解链曲线中可以了解到该曲线为较窄的单一峰，可以看出每个胃液标本的小RNA U6均被特异性扩增，没有引物二聚体和杂带的干扰。The method is basically the same as in Example 1, except that in step g, the amplified upstream primer is replaced by the small RNA U6 specific amplification upstream primer for piR-651, and the amplification curve of the expression level of small RNA U6 in the gastric juice is detected ( Figure 5) and melting curve (Figure 6), from the amplification curve, the Ct values of the two gastric juice samples detected are 29.60 and 32.04 respectively; from the melting curve, it can be known that the curve is a narrow single It can be seen that the small RNA U6 of each gastric juice sample was specifically amplified without the interference of primer dimers and heterogeneous bands.

实施例4Example 4

利用同一部位抽取的胃液标本中piRNA和小RNA U6的Ct值，就可根据公式 DCt = Ct_piRNA - Ct_U6计算出该piRNA的DCt值，根据DCt值的大小就可判断该piRNA的相对表达水平；DCt值小者，其对应piRNA的表达水平高；而DCt值大者，其对应piRNA的表达水平低。Using the Ct values of piRNA and small RNA U6 in gastric juice samples extracted from the same site, the DCt value of the piRNA can be calculated according to the formula DCt = Ct_piRNA - Ct_U6 , and the relative expression level of the piRNA can be judged according to the DCt value ; A small DCt value corresponds to a high expression level of piRNA; and a large DCt value corresponds to a low expression level of piRNA.

上述实施例中piR-651和piR-823也可以用其它piRNA特异性扩增上游引物代替，在此不一一列举；本发明建立了检测胃液中piRNA水平的方法，为研究胃组织的功能及其变化提供了依据。In the above examples, piR-651 and piR-823 can also be replaced by other piRNA-specific amplification upstream primers, which are not listed here; the present invention establishes a method for detecting piRNA levels in gastric juice, in order to study the function and function of gastric tissue. Its changes provide the basis.

Claims (3)

1. the detection method of piRNA in the gastric juice is characterized in that comprising the steps:

A, gastric juice extract and neutralization: extract gastric juice 5ml on an empty stomach with stomach tube, put into centrifuge tube, place on ice, add mass percentage concentration then and be 20% sodium hydroxide solution 0.5ml, put in the shaking table normal temperature jolting 20 minutes, with mass percentage concentration is that 20% sodium hydroxide solution is adjusted pH value to 7.0 ± 0.5, add mass percentage concentration again and be 1% trypsin solution 20 μ l, the whirlpool mixing was put in 37 ℃ of water baths digestion 15 minutes, under 4 ℃, centrifugal 20 minutes of 1000rpm;

B, RNA discharge: the supernatant liquor 200 μ l that get step a place the sterilization centrifuge tube, add the distilled water that the burnt ethylene carbonate of 50 μ l sterilization was handled, mixing, add Trizol solution 1ml again, blow and beat repeatedly 20 times, left standstill under the room temperature 10 minutes, under 4 ℃, centrifugal 15 minutes of 12000rpm;

C, chloroform extraction: the supernatant liquor of step b is moved in another sterilization centrifuge tube, add the 0.2ml chloroform, vortex oscillation 15 seconds, left standstill under the room temperature 5 minutes, under 4 ℃, centrifugal 15 minutes of 12000rpm draws upper water and moves into mutually in the sterilization centrifuge tube that coke acid second diester handled;

D, isopropanol precipitating: add the Virahol with described upper water equal volume in the sterilization centrifuge tube of step c, mixing left standstill under the room temperature 20 minutes, under 4 ℃, and centrifugal 15 minutes of 12000rpm, abandoning supernatant must precipitate;

E, washing with alcohol: in the precipitation of steps d, the adding mass percentage concentration is 75% ethanol 1ml, puts upside down 20 washings of mixing, under 4 ℃, centrifugal 3 minutes of 12000rpm abandons supernatant, again once with the same repeated washing of ethanol, under 4 ℃, centrifugal 3 minutes of 12000rpm abandons supernatant, drying is 10 minutes under the room temperature, add the dissolving of 15 μ l distilled waters, obtain the RNA extracting solution ,-80 ℃ of preservations are standby;

F, RNA reverse transcription: with miScript reverse transcription test kit the RNA extracting solution of step e is carried out reverse transcription reaction, obtain cDNA solution, it is standby to put-20 ℃ of preservations, and concrete operations are carried out according to this test kit specification sheets;

G, PCR detect: with miScript SYBR Green PCR detection kit the cDNA solution of step f is carried out pcr amplification reaction, the upstream primer of amplification is piRNA specific amplification upstream primer or little RNA U6 specific amplification upstream primer, the downstream primer of amplification is the general downstream primer of little RNA, detect with quantitative real time PCR Instrument, concrete operations are carried out according to detection kit and quantitative real time PCR Instrument specification sheets again.

2. the detection method of piRNA in a kind of gastric juice as claimed in claim 1 is characterized in that described piRNA specific amplification upstream primer is a has-piR-651 amplification upstream primer.

3. the detection method of piRNA in a kind of gastric juice as claimed in claim 1 is characterized in that described piRNA specific amplification upstream primer is a has-piR-823 amplification upstream primer.

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