技术领域technical field
本发明涉及生物技术领域，具体地，涉及一种在血浆和血清中检测miRNA的方法。The invention relates to the field of biotechnology, in particular to a method for detecting miRNA in plasma and serum.
背景技术Background technique
2008年Mitchell PS等发现血浆中miRNA能以稳定的形式存在，避免内源性RNA酶的降解。同年Shlomit G等证实miRNA可以在血浆和其他体液中稳定存在，并且表达谱随不同病理生理状态而改变。由于外周静脉血获取简单且病人容易接受，克服了样本量不足等困难，使得循环miRNA在疾病诊断中的应用研究成为研究的热点。In 2008, Mitchell PS et al. found that miRNA in plasma can exist in a stable form, avoiding the degradation of endogenous RNases. In the same year, Shlomit G et al confirmed that miRNAs can exist stably in plasma and other body fluids, and their expression profiles change with different pathophysiological states. Due to the simple acquisition of peripheral venous blood and easy acceptance by patients, it overcomes difficulties such as insufficient sample size, making the application of circulating miRNA in disease diagnosis a hot research topic.
最常用且有效的miRNA表达检测技术是荧光定量PCR法。目前通用步骤为：抽提样本RNA，逆转为miRNA cDNA，荧光定量检测miRNA表达水平。第一步抽提样本RNA较困难。由于血浆miRNA含量较低，若样本量较少时提取的RNA量不足后续试验。最常用的血浆循环RNA抽提法沿用传统TriZol抽提法：TriZol裂解，氯仿萃取，异丙醇沉淀，75％乙醇洗涤，DEPC水溶解。此方法提取效率低，对样本量要求大，而且有机试剂的残留会抑制后续PCR反应，造成miRNA检测效率下降。市场上的试剂盒抽提法为：裂解，萃取，磁珠或吸附柱吸附，洗涤，溶解。该方法通常会使用磁珠或吸附柱，会造成RNA的损失、破坏RNA的完整性，对样本需求量大而且费用较高。The most commonly used and effective miRNA expression detection technology is fluorescent quantitative PCR method. The current general steps are: extract sample RNA, reverse to miRNA cDNA, and detect miRNA expression level quantitatively by fluorescence. The first step is to extract sample RNA is more difficult. Due to the low plasma miRNA content, if the sample size is small, the amount of extracted RNA is insufficient for subsequent experiments. The most commonly used plasma circulating RNA extraction method follows the traditional TriZol extraction method: TriZol cleavage, chloroform extraction, isopropanol precipitation, 75% ethanol washing, and DEPC water dissolution. This method has low extraction efficiency and requires a large sample size, and the residue of organic reagents will inhibit the subsequent PCR reaction, resulting in a decrease in the detection efficiency of miRNA. The kit extraction methods on the market are: cracking, extraction, magnetic beads or adsorption column adsorption, washing, and dissolution. This method usually uses magnetic beads or adsorption columns, which will cause the loss of RNA, destroy the integrity of RNA, require a large amount of samples and be expensive.
并且，传统的血浆miRNA检测方法复杂，耗时间，成本高，重复性低，不利于临床推广和应用。Moreover, the traditional plasma miRNA detection method is complex, time-consuming, high cost, and low repeatability, which is not conducive to clinical promotion and application.
因此，本领域迫切需要开发一种简化检测方法、降低成本、提高重复性的在血浆和血清中检测miRNA的方法。Therefore, there is an urgent need in this field to develop a method for detecting miRNA in plasma and serum that simplifies the detection method, reduces the cost, and improves the repeatability.
发明内容Contents of the invention
本发明的目的是提供了一种简化检测方法、降低成本、提高重复性的在血浆和血清中检测miRNA的方法。The purpose of the present invention is to provide a method for detecting miRNA in plasma and serum which simplifies the detection method, reduces the cost and improves the repeatability.
本发明第一方面提供了一种非诊断性的免抽提的对测试样品进行miRNA检测的方法，所述方法包括步骤：The first aspect of the present invention provides a non-diagnostic, extraction-free method for miRNA detection of a test sample, the method comprising the steps of:
(i)提供一测试样品，所述测试样品为血浆和血清样品，将所述测试样品和预处理缓冲液混合，形成第一混合物，其中所述预处理缓冲液包括非离子型表面活性剂；(i) providing a test sample, the test sample is a plasma and serum sample, mixing the test sample and a pretreatment buffer to form a first mixture, wherein the pretreatment buffer includes a non-ionic surfactant;
(ii)将所述第一混合物进行加热处理，得到经处理的第二混合物；和(ii) subjecting said first mixture to heat treatment to obtain a treated second mixture; and
(iii)将所述第二混合物直接用作模板，检测所述测试样品中的miRNA的种类和/或含量。(iii) using the second mixture directly as a template to detect the type and/or content of miRNA in the test sample.
在另一优选例中，在步骤(i)中，所述预处理缓冲液包括一种或多种选自下组的非离子型表面活性剂：吐温20(TWEEN-20)、吐温21(TWEEN-21)、吐温40(TWEEN-40)、吐温60(TWEEN-60)、吐温61(TWEEN-61)、吐温80(TWEEN-80)、吐温81(TWEEN-81)、吐温85(TWEEN-85)、Triton X-100、NP-40、或其组合。In another preference, in step (i), the pretreatment buffer includes one or more nonionic surfactants selected from the group consisting of Tween 20 (TWEEN-20), Tween 21 (TWEEN-21), Tween 40 (TWEEN-40), Tween 60 (TWEEN-60), Tween 61 (TWEEN-61), Tween 80 (TWEEN-80), Tween 81 (TWEEN-81) , TWEEN-85, Triton X-100, NP-40, or a combination thereof.
在另一优选例中，所述非离子型表面活性剂包括吐温20(TWEEN-20)。In another preferred example, the nonionic surfactant includes Tween-20.
在另一优选例中，所述非离子型表面活性剂的浓度(V/V)为0.5-10％，较佳地，1-5％，更佳地，2-4％。In another preferred example, the concentration (V/V) of the nonionic surfactant is 0.5-10%, preferably 1-5%, more preferably 2-4%.
在另一优选例中，所用溶剂为水。In another preferred example, the solvent used is water.
在另一优选例中，所述非离子型表面活性剂的浓度为0.5-10wt％，较佳地，1-5wt％，更佳地，2-4wt％。In another preferred example, the concentration of the nonionic surfactant is 0.5-10 wt%, preferably 1-5 wt%, more preferably 2-4 wt%.
在另一优选例中，在步骤(i)中，所述预处理缓冲液的pH为6-9，较佳地，7-9，更佳地，7-8。In another preferred example, in step (i), the pH of the pretreatment buffer is 6-9, preferably 7-9, more preferably 7-8.
在另一优选例中，所述预处理缓冲液还包括Tris-HCl缓冲液。In another preferred example, the pretreatment buffer also includes Tris-HCl buffer.
在另一优选例中，所述Tris-HCl缓冲液的浓度为25-150mM，较佳地，30-100mM，更佳地，30-80mM。In another preferred example, the concentration of the Tris-HCl buffer is 25-150 mM, preferably 30-100 mM, more preferably 30-80 mM.
在另一优选例中，所述预处理缓冲液还包括金属螯合剂。In another preferred example, the pretreatment buffer also includes a metal chelating agent.
在另一优选例中，所述金属螯合剂选自下组：EDTA、EGTA、或其组合。In another preferred embodiment, the metal chelating agent is selected from the group consisting of EDTA, EGTA, or a combination thereof.
在另一优选例中，所述金属螯合剂包括EDTA。In another preferred example, the metal chelating agent includes EDTA.
在另一优选例中，所述金属螯合剂的浓度为0.5-2.0mM，较佳地，0.5-1.5mM，更佳地，0.8-1.2mM。In another preferred embodiment, the concentration of the metal chelating agent is 0.5-2.0 mM, preferably 0.5-1.5 mM, more preferably 0.8-1.2 mM.
在另一优选例中，在步骤(ii)中，所述加热温度为60-80℃，较佳地，70-75℃。In another preferred example, in step (ii), the heating temperature is 60-80°C, preferably 70-75°C.
在另一优选例中，在步骤(iii)中，用荧光定量PCR的方法检测测试样品中的miRNA的含量。In another preferred embodiment, in step (iii), the miRNA content in the test sample is detected by fluorescent quantitative PCR.
在另一优选例中，所述miRNA来自于哺乳动物，优选人。In another preferred example, the miRNA is from a mammal, preferably a human.
在另一优选例中，所述方法不包括抽提RNA的步骤。In another preferred example, the method does not include the step of extracting RNA.
在另一优选例中，所述方法为非治疗非诊断性的。In another preferred embodiment, the method is non-therapeutic and non-diagnostic.
在另一优选例中，所述方法检测所述测试样品中选自下组(A)的一个或多个miRNA的种类和/或数量：miR-20b、miR-106a、miR-16。In another preferred embodiment, the method detects the type and/or quantity of one or more miRNAs selected from the group (A): miR-20b, miR-106a, and miR-16 in the test sample.
在另一优选例中，所述方法检测所述测试样品中选自下组(B)的一个或多个miRNA的种类和/或数量：miR-671、miR-151、miR-433、miR-409。In another preferred example, the method detects the type and/or quantity of one or more miRNAs selected from the group (B) in the test sample: miR-671, miR-151, miR-433, miR- 409.
本发明第二方面提供了一种miRNA组合物，所述组合物包括如下miRNA或由如下miRNA构成：miR-20b、miR-106a、miR-16、miR-671、miR-151、miR-433、和miR-409。The second aspect of the present invention provides a miRNA composition, which includes or consists of the following miRNAs: miR-20b, miR-106a, miR-16, miR-671, miR-151, miR-433, and miR-409.
在另一优选例中，所述组合物包括选自下组(A)的至少一种或全部的miRNA：miR-20b、miR-106a、miR-16。In another preferred embodiment, the composition includes at least one or all miRNAs selected from the group (A): miR-20b, miR-106a, and miR-16.
在另一优选例中，所述组合物包括选自下组(B)的至少一种或全部的miRNA：miR-671、miR-151、miR-433、miR-409。In another preferred embodiment, the composition includes at least one or all miRNAs selected from the group (B): miR-671, miR-151, miR-433, and miR-409.
本发明第三方面提供了一种本发明第二方面所述组合物的用途，用于制备检测乳腺癌转移的诊断试剂或试剂盒。The third aspect of the present invention provides a use of the composition described in the second aspect of the present invention for preparing a diagnostic reagent or kit for detecting breast cancer metastasis.
在另一优选例中，所述组合物还被用作血浆中检测乳腺癌发生及转移的生物标志物。In another preferred example, the composition is also used as a biomarker in plasma to detect occurrence and metastasis of breast cancer.
在另一优选例中，所述试剂盒包括：(i)本发明第二方面所述的miRNA组合物作为对照品；和(ii)标签或说明书，其中，所述标签或说明书注明所述试剂盒用于检测或诊断乳腺癌转移。In another preferred example, the kit includes: (i) the miRNA composition described in the second aspect of the present invention as a reference; and (ii) a label or instructions, wherein the label or instructions indicate the The kit is used for detecting or diagnosing breast cancer metastasis.
在另一优选例中，所述诊断试剂是单克隆抗体。In another preferred example, the diagnostic reagent is a monoclonal antibody.
在另一优选例中，所述的检测乳腺癌转移是血浆检测。In another preferred example, the detection of breast cancer metastasis is plasma detection.
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, we will not repeat them here.
附图说明Description of drawings
图1显示了本发明的方法与传统方法的比较。Figure 1 shows a comparison of the method of the present invention with the conventional method.
图2显示了最佳方法的优选。其中，2A：三种不同处理样本方法的比较；2B：三种方法分别检测miR-16，显示M3(化学变性和加热变性相结合)的检测效率最高。Figure 2 shows the optimization of the best method. Among them, 2A: comparison of three different methods for processing samples; 2B: three methods for detecting miR-16 respectively, showing that M3 (combination of chemical denaturation and heating denaturation) has the highest detection efficiency.
图3显示了该检测方法的准确性。取不同体积的血浆样本，定量检测miR-16，其含量与样本体积呈正比例关系，其中，3A：利用M3(化学变性和加热变性相结合)，定量检测血浆miR-16；3B：miR-16扩增循环数；3C：将上述扩增循环数换算为miRNA相对表达量，和样本体积成线性正比关系。Figure 3 shows the accuracy of this detection method. Different volumes of plasma samples were taken to quantitatively detect miR-16, and its content was proportional to the sample volume, among which, 3A: use M3 (combined chemical denaturation and heat denaturation) to quantitatively detect plasma miR-16; 3B: miR-16 Amplification cycle number; 3C: The above-mentioned amplification cycle number is converted into the relative expression level of miRNA, which is linearly proportional to the sample volume.
图4显示了该检测方法的特异性。同一血浆样本中加入不同体积的外源性小RNA unisp6，利用该方法成功检测到unisp6，并且其检测到的含量与加入的量相符。miR-16作为内对照，其含量不随外源性unisp6的加入而改变。其中，4A：定量检测血浆中unisp6的丰度；4B：检测和unisp6无关的miR-16。Figure 4 shows the specificity of the assay. Different volumes of exogenous small RNA unisp6 were added to the same plasma sample, and unisp6 was successfully detected by this method, and the detected content was consistent with the added amount. miR-16 was used as an internal control, and its content did not change with the addition of exogenous unisp6. Among them, 4A: Quantitative detection of the abundance of unisp6 in plasma; 4B: Detection of miR-16 unrelated to unisp6.
图5通过检测miR-92a和let-7b，比较了传统的检测方法及本方案申请的检测方法。其中，5A显示血浆直接测定法(P)检测效率优于传统的RNA提取检测方法(R)；5B显示两种方法分别检测let-7b在不同样本中的差异，结果是一致的；5C显示两种方法分别检测miR-92a在不同样本中的差异，结果是一致的。Figure 5 compares the traditional detection method and the detection method of this application by detecting miR-92a and let-7b. Among them, 5A shows that the detection efficiency of plasma direct assay (P) is better than the traditional RNA extraction detection method (R); 5B shows that the two methods detect the difference of let-7b in different samples respectively, and the results are consistent; 5C shows that the two methods The two methods were used to detect the difference of miR-92a in different samples, and the results were consistent.
图6显示了血浆中miR-106a和miR-671可以作为乳腺癌转移的生物标志物。其中，6A显示4例正常人和6例乳腺癌患者(包括3例癌细胞非转移患者和3例癌细胞转移患者)血浆miRNA筛查，发现7个异常表达的miRNA；6B通过更多样本的验证，证实miR-106a在转移性乳腺癌血浆中显著性上调；6C通过更多样本的验证，证实miR-671在转移性乳腺癌血浆中显著性下调。Figure 6 shows that miR-106a and miR-671 in plasma can be used as biomarkers of breast cancer metastasis. Among them, 6A shows 4 cases of normal people and 6 cases of breast cancer patients (including 3 cases of non-metastatic cancer cells and 3 cases of cancer cell metastases) plasma miRNA screening, found 7 abnormally expressed miRNAs; 6B through more samples Verification, confirmed that miR-106a was significantly up-regulated in metastatic breast cancer plasma; 6C, through verification of more samples, confirmed that miR-671 was significantly down-regulated in metastatic breast cancer plasma.
图7显示加热处理的样本，利用血浆直接测定法，能够检测到miR-16，而SDS处理的样本，利用血浆直接测定法未能检测到miR-16。Figure 7 shows that heat-treated samples could detect miR-16 by direct plasma assay, while SDS-treated samples could not detect miR-16 by direct plasma assay.
图8显示加热处理的样本，利用血浆直接测定法，能够检测到miR-21，而SDS处理的样本，利用血浆直接测定法未能检测到miR-21。Figure 8 shows that heat-treated samples could detect miR-21 by direct plasma assay, while SDS-treated samples could not detect miR-21 by direct plasma assay.
图9显示加热处理的样本，利用血浆直接测定法，能够检测到5s rRNA，而SDS处理的样本，利用血浆直接测定法未能检测到5s rRNA。Figure 9 shows that 5s rRNA can be detected by direct plasma assay in heat-treated samples, while 5s rRNA cannot be detected by direct plasma assay in SDS-treated samples.
具体实施方式detailed description
本发明人经过广泛而深入的研究，首次开发了一种新型的检测miRNA的方法，该方法将血浆和血清样品进行加热和非离子表面活性剂处理，所得到的混合物不经RNA提取直接用作模板，用于检测血浆和血清中miRNA的含量。该检测方法的检测结果具有高准确性、高灵敏度、高特异性、高检测效率的特点。此外，本发明人还发现一系列miRNA分子，可作为诊断乳腺癌转移的标记物。在此基础上，发明人完成了本发明。After extensive and in-depth research, the inventors have developed a novel method for detecting miRNA for the first time. In this method, plasma and serum samples are heated and treated with non-ionic surfactants, and the resulting mixture is used directly without RNA extraction. Template for detection of miRNA levels in plasma and serum. The detection result of the detection method has the characteristics of high accuracy, high sensitivity, high specificity and high detection efficiency. In addition, the inventors also found a series of miRNA molecules, which can be used as markers for diagnosing breast cancer metastasis. On this basis, the inventors have completed the present invention.
如本文所用，术语“预处理缓冲液”、“变性缓冲液”可互换使用，均指一种含有吐温20(0.5-10％V/V)、EDTA(0.8-1.2mM)和Tris-HCl(30-80mM)的溶液。As used herein, the terms "pretreatment buffer" and "denaturation buffer" are used interchangeably, and both refer to a buffer containing Tween 20 (0.5-10% V/V), EDTA (0.8-1.2 mM) and Tris- A solution of HCl (30-80 mM).
检测方法Detection method
本发明提供了一种可用于检测微小RNA(miRNA)的新方法。The present invention provides a new method that can be used to detect microRNA (miRNA).
本发明检测方法采用了对血浆和血清进行加热和变性缓冲液(包括非离子型表面活性剂)共处理，所得到的混合物不经RNA提取步骤，直接用于miRNA的检测。The detection method of the present invention adopts the co-treatment of plasma and serum by heating and denaturing buffer (including non-ionic surfactant), and the obtained mixture is directly used for the detection of miRNA without the RNA extraction step.
本发明的方法与传统方法的比较如图1所示。在本发明中，一个典型的miRNA检测方法包括如下基本步骤：The method of the present invention is compared with traditional method as shown in Figure 1. In the present invention, a typical miRNA detection method comprises the following basic steps:
(i)提供一测试样品，所述测试样品为血浆和血清样品，将所述测试样品和预处理缓冲液混合，形成第一混合物，其中所述预处理缓冲液包括非离子型表面活性剂；(i) providing a test sample, the test sample is a plasma and serum sample, mixing the test sample and a pretreatment buffer to form a first mixture, wherein the pretreatment buffer includes a non-ionic surfactant;
(ii)将所述第一混合物进行加热处理，得到经处理的第二混合物；和(ii) subjecting said first mixture to heat treatment to obtain a treated second mixture; and
(iii)将所述第二混合物直接用作模板，检测所述测试样品中的miRNA的种类和/或含量。(iii) using the second mixture directly as a template to detect the type and/or content of miRNA in the test sample.
在本发明中，一种优选的检测方法的步骤包括：In the present invention, the steps of a preferred detection method include:
(1)准备变性缓冲液：2.5％(v/v)Tween 20,50mmol/L Tris-HCl,1mmol/L EDTA(1) Prepare denaturation buffer: 2.5% (v/v) Tween 20, 50mmol/L Tris-HCl, 1mmol/L EDTA
(2)5uL血浆加5uL等体积变性缓冲液，反应10分钟，75℃水浴5min，入冰冷却，离心。(2) Add 5uL plasma with 5uL equal volume of denaturing buffer, react for 10 minutes, bathe in 75°C water for 5 minutes, cool in ice, and centrifuge.
(3)利用常规市售miRNA逆转录试剂盒，将miRNA反转录为cDNA，稀释10-100倍备用。(3) Using a conventional commercially available miRNA reverse transcription kit, the miRNA is reverse-transcribed into cDNA and diluted 10-100 times for later use.
(4)荧光定量PCR反应，扩增miRNA，检测miRNA在血浆样本的表达丰度。(4) Fluorescent quantitative PCR reaction, amplifying miRNA, and detecting the expression abundance of miRNA in plasma samples.
16uL反应体系：1uL cDNA，1uL待测miRNA前引物(2.5pmol/uL)，1uL通用后引物(2.5pmol/uL)，8uL SYBR荧光试剂，5uL H2O。16uL reaction system: 1uL cDNA, 1uL pre-primer for the miRNA to be tested (2.5pmol/uL), 1uL universal post-primer (2.5pmol/uL), 8uL SYBR fluorescent reagent, 5uL H2 O.
反应条件：50℃、2min；95℃、10min；40个循环：95℃、15s，60℃、1min。Reaction conditions: 50°C, 2min; 95°C, 10min; 40 cycles: 95°C, 15s, 60°C, 1min.
本发明的检测方法不仅省略了RNA的提取步骤，还具有检测结果高准确性、高特异性、高灵敏度、高检测效率的特点。The detection method of the present invention not only omits the RNA extraction step, but also has the characteristics of high accuracy, high specificity, high sensitivity and high detection efficiency of detection results.
检测乳腺癌发生及转移的生物标志物Detection of biomarkers of breast cancer development and metastasis
本发明还提供了miRNA组合物，可被用作检测血浆中乳腺癌发生及转移的生物标志物。The invention also provides the miRNA composition, which can be used as a biomarker for detecting the occurrence and metastasis of breast cancer in plasma.
在本发明中，本发明的miRNA组合物选自下组：miR-20b、miR-106a、miR-16、miR-671、miR-151、miR-433、miR-409、或其组合。In the present invention, the miRNA composition of the present invention is selected from the group consisting of miR-20b, miR-106a, miR-16, miR-671, miR-151, miR-433, miR-409, or a combination thereof.
其中，miR-20b、miR-106a和miR-16在转移性乳腺癌患者的血浆中能够特异性高表达；miR-671、miR-151、miR-433和miR-409在转移性乳腺癌患者的血浆中能够特异性低表达。Among them, miR-20b, miR-106a and miR-16 can specifically and highly express in the plasma of patients with metastatic breast cancer; It can be specifically and lowly expressed in plasma.
本发明的优点主要包括：Advantage of the present invention mainly comprises:
(1)该检测方法省略了RNA的提取步骤，方法简单，成本更低。(1) The detection method omits the RNA extraction step, the method is simple, and the cost is lower.
(2)该检测方法的检测结果具有高准确性、高特异性、高检测效率的特点。(2) The detection result of the detection method has the characteristics of high accuracy, high specificity and high detection efficiency.
(3)该检测方法灵敏度更高，避免了血浆RNA制备过程中的miRNA损失。(3) The detection method has higher sensitivity and avoids the loss of miRNA during the preparation of plasma RNA.
(4)该检测方法快捷便利，和传统方法相比，检测时间缩短了一半。(4) The detection method is fast and convenient, and compared with the traditional method, the detection time is shortened by half.
(5)该检测方法避免使用Trizol等有机溶剂，排除了传统方法残存有机溶剂对后续检测的影响。(5) This detection method avoids the use of organic solvents such as Trizol, and eliminates the influence of residual organic solvents in traditional methods on subsequent detection.
(6)基于上述优点，该检测方法更适于临床检验和检测。(6) Based on the above advantages, the detection method is more suitable for clinical examination and detection.
(7)首次发现，本发明的miRNA组合物可以被用作检测血浆中乳腺癌发生及转移的生物标志物。(7) It is found for the first time that the miRNA composition of the present invention can be used as a biomarker for detecting the occurrence and metastasis of breast cancer in plasma.
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量或体积百分比和重量份数。Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate specific condition in the following examples is generally according to conventional conditions, such as Sambrook et al., molecular cloning: the conditions described in the laboratory handbook (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacture conditions recommended by the manufacturer. Percentages and parts are by weight or volume unless otherwise indicated.
1.方法1. Method
(1)准备变性缓冲液：2.5％(v/v)Tween 20,50mmol/L Tris-HCl,1mmol/L EDTA(1) Prepare denaturation buffer: 2.5% (v/v) Tween 20, 50mmol/L Tris-HCl, 1mmol/L EDTA
(2)5uL血浆加5uL等体积变性缓冲液，反应10分钟，75℃水浴5min，入冰冷却，离心。(2) Add 5uL plasma with 5uL equal volume of denaturing buffer, react for 10 minutes, bathe in 75°C water for 5 minutes, cool in ice, and centrifuge.
(3)利用常规市售miRNA逆转录试剂盒，将miRNA反转录为cDNA，稀释10-100倍备用。(3) Using a conventional commercially available miRNA reverse transcription kit, the miRNA is reverse-transcribed into cDNA and diluted 10-100 times for later use.
(4)荧光定量PCR反应，扩增miRNA，检测miRNA在血浆样本的表达丰度。(4) Fluorescent quantitative PCR reaction, amplifying miRNA, and detecting the expression abundance of miRNA in plasma samples.
16uL反应体系：1uL cDNA，1uL待测miRNA前引物(2.5pmol/uL)，1uL通用后引物(2.5pmol/uL)，8uL SYBR荧光试剂，5uL H2O。16uL reaction system: 1uL cDNA, 1uL pre-primer for the miRNA to be tested (2.5pmol/uL), 1uL universal post-primer (2.5pmol/uL), 8uL SYBR fluorescent reagent, 5uL H2O.
反应条件：50℃、2min；95℃、10min；40个循环：95℃、15second，60℃、1min。Reaction conditions: 50°C, 2min; 95°C, 10min; 40 cycles: 95°C, 15second, 60°C, 1min.
2.优化的方法2. Optimization method
首先比较了三种不同的蛋白变性方法，M1是加热变性，M2是变性缓冲液化学变性，M3是把化学变性和加热变性结合一起，比较不同方法对miRNA在血浆中的释放和定量检测的影响。Firstly, three different protein denaturation methods were compared, M1 is heat denaturation, M2 is chemical denaturation of denaturation buffer, M3 is a combination of chemical denaturation and heat denaturation, and compared the effects of different methods on the release and quantitative detection of miRNA in plasma .
利用上述三种不同的方法，定量检测了同一个血浆样本中miR-16的表达丰度，结果如图2A和2B所示。结果显示，M3检测效率最高(循环数最小)，M1次之，M2效率最低(循环数最大)。Using the above three different methods, the expression abundance of miR-16 in the same plasma sample was quantitatively detected, and the results are shown in Figure 2A and 2B. The results showed that M3 had the highest detection efficiency (minimum cycle number), followed by M1, and M2 had the lowest detection efficiency (maximum cycle number).
结果表明，利用血浆蛋白变性直接检测miRNA方法可行，并且M3方法(化学变性和加热变性结合)对于血浆miRNA检测效率最高。The results showed that the direct detection of miRNA by plasma protein denaturation was feasible, and the M3 method (combination of chemical denaturation and heat denaturation) was the most efficient for the detection of plasma miRNA.
3.方法准确性的验证3. Validation of method accuracy
分别取1、4、8ul血浆，利用M3所述方法，定量检测血浆miR-16，以验证不同体积血浆中miR-16量的不同。1, 4, and 8 ul of plasma were collected, and the method described in M3 was used to quantitatively detect plasma miR-16 to verify the difference in the amount of miR-16 in different volumes of plasma.
结果如图3A、3B、3C所示。结果显示，1、4、8ul血浆分别对应的miR-16扩增循环数为31.8、29.6、28.5。血浆体积越大，循环数越小，符合预期。将上述扩增循环数换算为miRNA相对表达量，和样本体积成线性正比关系，R2达到0.999，结果表明，本发明的检测方法对血浆miRNA的检测具有高准确性。The results are shown in Figures 3A, 3B, and 3C. The results showed that the number of cycles of miR-16 amplification corresponding to 1, 4, and 8ul plasma was 31.8, 29.6, and 28.5, respectively. Larger plasma volumes were associated with lower cycle numbers, as expected. The above-mentioned number of amplification cycles is converted into the relative expression of miRNA, which is linearly proportional to the sample volume, and R2 reaches 0.999.The results show that the detection method of the present invention has high accuracy for the detection of plasma miRNA.
4.方法特异性的验证4. Validation of method specificity
在一个血浆样本中，加入一种外源性的RNA(unisp6)。此RNA在血浆中本身不存在。设定三组实验，等量血浆中分别给予0、1、4ul的unisp6，再利用M3所述方法，定量检测血浆中unisp6的丰度，以验证该方法对某个特定miRNA检测的特异性。结果如图4A所示。结果显示，unisp6在0ul的实验组中检测不到；而在1ul和4ul的实验组中能够检测到，并且其丰度相对比值≈4，和unisp6加入量匹配，结果证明了该方法对RNA定量分析具有高特异性。In a plasma sample, an exogenous RNA (unisp6) was added. This RNA itself does not exist in plasma. Three sets of experiments were set up, 0, 1, and 4ul of unisp6 were administered to equal amounts of plasma, and then the method described in M3 was used to quantitatively detect the abundance of unisp6 in plasma to verify the specificity of this method for the detection of a specific miRNA. The results are shown in Figure 4A. The results showed that unisp6 could not be detected in the 0ul experimental group; it could be detected in the 1ul and 4ul experimental groups, and its relative abundance ratio was ≈4, which matched the amount of unisp6 added. The results proved that this method can quantify RNA The analysis is highly specific.
在上述体系中，检测和unisp6无关的miR-16，结果如图4B所示。结果表明，三组体系中miR-16的表达丰度一致，进一步验证了该方法对RNA定量分析具有高特异性。In the above system, miR-16, which is unrelated to unisp6, was detected, and the results are shown in Figure 4B. The results showed that the expression abundance of miR-16 in the three systems was consistent, further verifying that the method has high specificity for RNA quantitative analysis.
5.检测效率的验证5. Verification of detection efficiency
设计一组实验，比较传统提取RNA的方法和本发明的方法，用于同一个血浆样本miRNA检测，通过cDNA不同稀释倍数，取相当于源自0.02ul血浆的两种方法的cDNA，分别检测miRNA let-7b和miR-92a。结果如图5A所示。结果显示，与传统的分析方法相比，本发明的方法具有更高的检测效率，ΔCt值相差0.7～1.1，相当于检测效率高一倍左右。Design a group of experiments, compare the traditional method of extracting RNA and the method of the present invention, for the same plasma sample miRNA detection, through different dilution times of cDNA, take the cDNA equivalent to the two methods derived from 0.02ul plasma, and detect miRNA respectively let-7b and miR-92a. The results are shown in Figure 5A. The results show that, compared with the traditional analysis method, the method of the present invention has higher detection efficiency, and the difference of ΔCt value is 0.7-1.1, which is equivalent to about twice the detection efficiency.
设计另外一组实验，比较传统提取RNA的方法和本发明的方法，用于检测两种不同血浆中某miRNA的表达差异性。发明人用两种方法，分别检测了样本S4和S5中let-7b和miR-92a的表达。结果如图5B和5C所示。结果显示，两种方法均检测到样本S4和S5的let-7b，ΔCt≈3，相当于S4的let-7b比S5高8倍，并且两种方法均检测到样本S4和S5的mIR-92a，ΔCt≈2，相当于S4的let-7b比S5高4倍。该实验进一步证明了本发明的方法的检测效率非常高。Another set of experiments was designed to compare the traditional method of extracting RNA with the method of the present invention, and was used to detect the expression difference of a certain miRNA in two different plasmas. The inventors used two methods to detect the expression of let-7b and miR-92a in samples S4 and S5, respectively. The results are shown in Figures 5B and 5C. The results showed that both methods detected let-7b of samples S4 and S5, ΔCt≈3, equivalent to 8 times higher let-7b of S4 than S5, and both methods detected mIR-92a of samples S4 and S5 , ΔCt≈2, which corresponds to 4 times higher let-7b in S4 than in S5. This experiment further proves that the detection efficiency of the method of the present invention is very high.
6.血清中检测miRNA6. Detection of miRNA in serum
收集外周血到未加抗凝剂的采血管中，室温静置30分钟,1500rpm离心5分钟，收集上清，得到待测血清样本。Collect peripheral blood into a blood collection tube without anticoagulant, let it stand at room temperature for 30 minutes, centrifuge at 1500 rpm for 5 minutes, collect the supernatant, and obtain the serum sample to be tested.
用上述方法(1和2)分别对血清样本中的miRNA(如miR-16)进行检测。Use the above methods (1 and 2) to detect miRNA (such as miR-16) in serum samples respectively.
结果显示，M3方法(化学变性和加热变性结合)能够成功检测到血清miRNA,但对于同一血液样本，血浆miRNA含量高于血清miRNA含量(C1：C2＞130％)，可能归因于血清制备过程中部分miRNA随结合蛋白一起沉淀损失。The results showed that the M3 method (combination of chemical denaturation and heat denaturation) could successfully detect serum miRNA, but for the same blood sample, the plasma miRNA content was higher than the serum miRNA content (C1:C2>130%), which may be attributed to the serum preparation process Some miRNAs were lost by precipitation along with binding proteins.
结果表明，可直接检测血清的miRNA，并且M3方法(化学变性和加热变性结合)对于血浆miRNA检测效率最高。The results showed that serum miRNA can be detected directly, and the M3 method (combination of chemical denaturation and heat denaturation) has the highest detection efficiency for plasma miRNA.
7.临床应用7. Clinical application
之前通过miRNA基因芯片技术，分析了六例乳腺癌患者血浆样本及四例年龄匹配的健康女性血浆样本，其中患者三例为非转移乳腺癌、另三例为发生转移的乳腺癌，找到了三个在转移性乳腺癌患者血浆特异高表达的miRNA(miR-20b,miR-106a,miR-16)，及四个在转移性乳腺癌患者血浆特异低表达的miRNA(miR-671，miR-151，miR-433，miR-409)。如图6A、6B和6C所示。Previously, the plasma samples of six breast cancer patients and four age-matched healthy women were analyzed by miRNA gene chip technology, among which three patients had non-metastatic breast cancer and the other three had metastatic breast cancer, and found three One miRNA (miR-20b, miR-106a, miR-16) specifically highly expressed in the plasma of patients with metastatic breast cancer, and four miRNAs (miR-671, miR-151) specifically low expressed in the plasma of patients with metastatic breast cancer , miR-433, miR-409). As shown in Figures 6A, 6B and 6C.
利用本发明的血浆直接测定法M3(化学变性和加热变性相结合)，重新收集了三例非转移乳腺癌患者血浆、三例发生转移的乳腺癌患者血浆、三例健康女性的血浆，对上述miRNA分别进行了验证，发现miR-106a在转移性乳腺癌患者血浆特异高表达，miR-671在转移性乳腺癌患者血浆特异低表达。这两个结果与之前的基因芯片检测结果一致，说明通过不同样本，不同方法的分析，确认了这两个miRNA可以作为转移性乳腺癌患者血浆生物标志物，具有临床应用价值和潜力，可用于乳腺癌转移的早期诊断。Utilize plasma direct determination method M3 of the present invention (chemical denaturation and thermal denaturation are combined), collected three cases of non-metastatic breast cancer patient plasma, three cases of metastatic breast cancer patient plasma, three cases of healthy women's plasma, the above-mentioned The miRNAs were verified separately, and it was found that miR-106a was specifically highly expressed in the plasma of metastatic breast cancer patients, and miR-671 was specifically and lowly expressed in the plasma of metastatic breast cancer patients. These two results are consistent with the previous gene chip detection results, indicating that through the analysis of different samples and different methods, it has been confirmed that these two miRNAs can be used as plasma biomarkers in patients with metastatic breast cancer, which have clinical application value and potential, and can be used for Early diagnosis of breast cancer metastasis.
对比例1Comparative example 1
方法同1，区别在于，用SDS替代变性缓冲液，取5ul血浆，分别用加热处理或者等体积10％SDS处理，然后利用本发明的miRNA直接检测法，对三个微小RNA(如miR-16、miR-21、5srRNA)进行检测,结果如图7－9所示。The method is the same as 1, the difference is that the denaturing buffer is replaced with SDS, 5ul of plasma is taken, and treated with heat treatment or an equal volume of 10% SDS respectively, and then using the miRNA direct detection method of the present invention to detect three microRNAs (such as miR-16 , miR-21, 5srRNA) were detected, and the results are shown in Figure 7-9.
结果如图7-9所示。结果显示，加热变性处理的样本，扩增良好，利用血浆直接测定法，能够检测到miR-16、miR-21和5s rRNA，而SDS变性处理的样本，没有扩增曲线，利用血浆直接测定法均未能检测到上述miRNA。The results are shown in Figure 7-9. The results showed that heat-denatured samples had good amplification, and miR-16, miR-21 and 5s rRNA could be detected by direct plasma assay, while SDS denatured samples had no amplification curve, and direct plasma assay could detect miR-16, miR-21 and 5s rRNA. None of the above miRNAs could be detected.
结果表明，用SDS处理，无法对三个微小RNA(如miR-16、miR-21、5s rRNA)进行检测。The results showed that three microRNAs (such as miR-16, miR-21, 5s rRNA) could not be detected by SDS treatment.
对比例2Comparative example 2
方法同1，区别在于，用SDS替代变性缓冲液，对血浆进行加热和等体积10％SDS处理，然后利用本发明的miRNA直接检测法，对三个微小RNA(如miR-16、miR-21、5s rRNA)进行检测，结果显示，用加热和SDS同时处理，没有扩增曲线。The method is the same as 1, the difference is that the denaturing buffer is replaced by SDS, the plasma is heated and treated with an equal volume of 10% SDS, and then the miRNA direct detection method of the present invention is used to detect three microRNAs (such as miR-16, miR-21 , 5s rRNA) were detected, and the results showed that there was no amplification curve when heating and SDS were processed simultaneously.
结果表明，对血浆同时进行加热和SDS处理，无法对三个微小RNA(如miR-16、miR-21、5s rRNA)进行检测。The results showed that three microRNAs (such as miR-16, miR-21, 5s rRNA) could not be detected by simultaneous heating and SDS treatment of plasma.
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.
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| CN201610061103.8ACN107012199A (en) | 2016-01-28 | 2016-01-28 | A kind of method that miRNA is detected in blood plasma and serum |
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