WO2012142924A1 - 检测miRNA的方法及引物及其应用 - Google Patents

检测miRNA的方法及引物及其应用 Download PDF

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WO2012142924A1
WO2012142924A1 PCT/CN2012/073976 CN2012073976W WO2012142924A1 WO 2012142924 A1 WO2012142924 A1 WO 2012142924A1 CN 2012073976 W CN2012073976 W CN 2012073976W WO 2012142924 A1 WO2012142924 A1 WO 2012142924A1
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mirna
primer
oligo
primers
sequence
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PCT/CN2012/073976
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French (fr)
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苟德明
康康
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深圳大学
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Priority to EP12774868.9A priority Critical patent/EP2700719A4/en
Publication of WO2012142924A1 publication Critical patent/WO2012142924A1/zh
Priority to US14/056,919 priority patent/US20140045188A1/en

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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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • 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
    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates

Definitions

  • the invention relates to the field of biomedicine, in particular to a method for detecting miRNA and a primer and the application thereof in early diagnosis and prognosis of a major disease such as a tumor. Background technique
  • MicroRNAs are a class of non-coding single-stranded small RNAs of about 22 nucleotides in length, which are widely found in eukaryotes such as animals, plants, and nematodes. By specifically binding to the target mRNA 3, the non-coding region (3, UTR), the miRNA degrades the target mRNA or represses its post-transcriptional translation, ultimately resulting in a decrease in protein synthesis of the target gene. miRNAs are involved in all levels of life activity, including embryonic development, organ formation, cell proliferation, apoptosis, cellular stress response, stem cell differentiation, endocrine regulation, immune regulation, and disease development and progression.
  • miRNAs will have great potential as new biomarkers for early stages of major diseases such as cancer. Diagnosis can be used as a new target for gene drugs. Obviously, in order to use miRNAs for early diagnosis of diseases such as cancer, a method for rapid, sensitive, and specific quantitative detection of miRNA expression is needed.
  • Mature miRNAs have small fragments, no poly(A) tails, small sequence differences between different miRNAs (some have only one nucleotide difference), and the expression levels in cells are generally low, which all give quantitative detection of miRNAs. Work brings difficulties and challenges. Despite this, scientists have developed a variety of miRNA detection methods, which can be broadly classified into two categories: one is probe direct hybridization without sample amplification; the other is PCR-based miRNA detection.
  • the miRNA detection method based on probe hybridization technology is a direct detection method, and does not require pre-amplification of sample RNA, mainly as follows: Northern blot technology, biochip technology and microsphere-based flow cytometry.
  • the basic principle of these techniques is to hybridize the probe to the RNA sample and then perform signal detection.
  • Northern blot technology as a classic method for detecting RNA, Often used to evaluate the reliability of other methods; however, this technique requires a large amount of sample, which is relatively time consuming and labor intensive, and is not suitable for high throughput analysis.
  • Biochip technology enables high-throughput analysis of miRNAs, ie simultaneous detection of multiple miRNAs on a single chip; but the disadvantages are low accuracy, poor repeatability, and expensive experiments.
  • Microsphere-based flow cytometry technology immobilizes the probe on the microspheres and in the liquid phase, which is more conducive to capture miRNA sequences, thus improving accuracy; however, the technique requires the use of special flow cytometry and micro The ball, the high cost of the experiment, is also not conducive to promotion.
  • the poly(A) polymerase tailing method first uses a poly(A) polymerase to bring a poly(A) tail to the 3' end of the miRNA, and then reverses it with an Oligo (dT) primer containing a linker sequence at the 5' end. Recording, adding a linker to the first strand cDNA, providing a reverse universal primer sequence for subsequent PCR amplification, and then using a miRNA sequence-specific forward primer to achieve PCR amplification.
  • the advantage of this method is that the cartridge, rapid, and reverse transcription primers are versatile for miRNAs, so the detection cost is low; however, the use of universal reverse transcription primers reduces the specificity and sensitivity of the assay.
  • Stem ring primer method Stem ring primer method
  • the stem-loop primer method is generally more specific than the poly(A) polymer alcohol tail-tailing method; however, because the method uses a sequence-specific probe, for Qualcomm The amount of miRNA analysis is too expensive.
  • S-Oligo(dT) primer for quantitative detection of miRNA wherein the S-Oligo(dT) primer starts from the 5th end by a PCR universal primer sequence, a universal probe sequence, Oligo (dT) and The specific sequence of the 3, terminal nucleotide complementary pairing of the miRNA molecule is composed of 4 parts.
  • the S-Oligo (dT) primer for quantitatively detecting a miRNA wherein the S-Oligo (dT) primer is a 14-20 base PCR universal primer sequence from the 5th end, 14 to 20 A universal base probe sequence of 8 to 30 dTs and a specific sequence complementary to the 3, 3, and 8 nucleotides of the miRNA molecule of interest.
  • the S-Oligo (dT) primer for quantitatively detecting a miRNA wherein the primer is a 16-base PCR universal primer sequence and a 17-base universal probe sequence from the 5th end, 11 dT and a specific sequence complementary to the 3, 6 nucleotides of the miRNA molecule of interest.
  • a method for quantitatively detecting a specific miRNA using the above-described primer for quantitatively detecting a miRNA comprising the following steps:
  • Design of S100, S-Oligo(dT) primers and specific upstream primers for miRNA Design S-Oligo (dT) primers and specific upstream primers for miRNA based on sequence information of the target miRNA;
  • RNA tailing tailing the total RNA with PolyA polymerase to obtain RNA-PolyA;
  • Real-time PCR quantitative detection of S500 and miRNA Real-time PCR was performed using miRNA specific primers and downstream universal primers using the first strand cDNA of miRNA as a template, and then the PCR results were analyzed.
  • the method for quantitatively detecting miRNA wherein the miRNA specific upstream primer is a miRNA specific sequence containing no 3, 3 to 8 bases, and a downstream universal primer of the miRNA is derived from a S-Oligo (dT) primer.
  • the method for quantitatively detecting miRNA wherein the real-time PCR quantitative detection in step S500 is performed by a probe method or a SYBR fluorescent dye method; the probe used in the probe method is a universal probe, and the sequence thereof is from A universal probe sequence of 14-20 bases on the S-Oligo (dT) primer.
  • a method for quantitatively detecting different miRNAs using the above-described primers for quantitatively detecting miRNAs comprising the following steps:
  • Design of specific upstream primers for S100, S-Oligo(dT) primers and miRNAs Design different S-Oligo(dT) primers and specific upstream primers for miRNA based on sequence information of different target miRNAs;
  • RNA extraction lysis of cells, extraction of total RNA, and identification of RN A purity and integrity identification and determination of RNA concentration;
  • RNA tailing tailing the total RNA with PolyA polymerase to obtain RNA-PolyA;
  • Real-time PCR quantitative detection of S500 and miRNA Real-time PCR was performed using the miRNA specific first primer and the downstream universal primers as the template, and then the PCR results were analyzed.
  • the method for quantitatively detecting miRNA wherein the miRNA specific upstream primer is a miRNA specific sequence containing no 3, 3 to 8 bases, and a downstream universal primer of the miRNA is derived from a S-Oligo (dT) primer.
  • the method for quantitatively detecting miRNA wherein the real-time PCR quantitative detection in step S500 is performed by a probe method or a SYBR fluorescent dye method; the probe used in the probe method is a universal probe, and the sequence thereof is from A universal probe sequence of 14-20 bases on the S-Oligo (dT) primer.
  • the above method for quantitatively detecting a miRNA wherein the method for quantitatively detecting miRNA is applied to early diagnosis and prognosis of a major disease such as a tumor.
  • the method is characterized by significantly higher sensitivity and specificity than traditional methods, high-throughput analysis, single operation, rapid, low cost, and can be widely applied to early diagnosis and prognosis of major diseases such as tumors.
  • Figure 1 is a schematic diagram of the S-Oligo (dT) method for quantitative detection of miRNAs of the present invention.
  • Fig. 2 is a graph showing the amplification of hsa-miR-21 miRNA in female cervical cancer Hda cells by the S-Oligo (dT) method of the present invention.
  • Fig. 3 is a standard curve showing the amplification of hsa-miR-21 miRNA in female cervical cancer Hda cells by the S-Oligo (dT) method of the present invention.
  • Figure 4 is a graph showing the sensitivity comparison of different real-time RT-PCR amplifications of hsa-miR-21 in the examples of the present invention.
  • the present invention provides a method for detecting miRNA, a primer and an application thereof, and the present invention will be further described in detail below in order to clarify and clarify the objects, technical solutions and effects of the present invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
  • the miRNA reverse transcription primer for detecting miRNA provided by the present invention, S-Oligo (dT) primer, starts from the 5th end by PCR universal primer, Universal probe, 01igo (dT) and a specific base of four complementary nucleotide pairs of the target miRNA molecule.
  • the S-Oligo(dT) primer for quantitative detection of miRNA is a 14-20 base PCR universal primer sequence, a 14-20 base universal probe sequence, 8 ⁇ 30 from the 5th end.
  • the present invention also provides a novel method for quantitative detection of miRNA, the S-Oligo (dT) method.
  • S-Oligo (dT) method uses S-Oligo (dT) primers for reverse transcription of specific miRNAs, or different S-Oligo (dT) primers to simultaneously reverse-transcribe different miRNA samples, and then use each miRNA
  • the specific upstream primers and the universal downstream primers were used for real-time PCR quantitative detection of the corresponding miRNAs.
  • miRNA-specific upstream primers are miRNAs that do not contain 3, 3 to 8 bases.
  • the heterologous sequence, the universal primer for the downstream of the miRNA is derived from the universal primer sequence of 14-20 bases of the S-Oligo (dT) primer.
  • the detection of the PCR product can be performed by either a fluorescent dye method or a probe method.
  • the probe method uses a universal probe whose sequence is derived from a universal probe sequence of 14-20 bases on the S-Oligo (dT) primer.
  • the S-Oligo (dT) primer provided in the embodiment of the present invention has the following composition: 16 primers universal PCR primer sequence, 17 base universal probe sequence, 11 primers from the end of the primer 5 dT (Oligo(dT)) and a specific sequence complementary to the 3, 6 nucleotides of the miRNA molecule of interest.
  • the miRNA-specific upstream primer is a miRNA-specific sequence that does not contain a 3, 6-base miRNA
  • the downstream universal primer for miRNA is derived from the 16-base universal primer sequence of the S-Oligo (dT) primer.
  • the universal probe used has a sequence derived from a 17 base universal probe sequence on a S-Oligo (dT) primer.
  • the S-Oligo (dT) method for quantitative detection of miRNA provided by the present invention can be applied to early diagnosis and prediction of major diseases such as tumors.
  • the specific steps of the miRNA quantitative detection method of the present invention are as follows:
  • Design of S100, S-Oligo(dT) primers and specific upstream primers for miRNA Design S-Oligo (dT) primers and specific upstream primers for miRNA based on sequence information of the target miRNA;
  • RNA extraction lysis of cells, extraction of total RNA, identification of RN A purity and integrity, determination of RNA concentration;
  • RNA tailing tailing the total RNA with PolyA polymerase to obtain RNA-PolyA;
  • Real-time PCR quantitative detection of S500 and miRNA Real-time PCR was performed using miRNA specific primers and downstream universal primers using the first strand cDNA of miRNA as a template, and then the PCR results were analyzed.
  • S-Oligo (dT) assay detects significant increases in miRNA specificity:
  • the number of bases complementary to the target miRNA by the S-Oligo(dT) primer is 17 bases (6 specific bases and 11 dT), which is significantly superior to the poly(A) polymerized alcohol in anchoring strength to the target miRNA.
  • Tail method only Oligo (dT)
  • stem loop primer method only 6 specific bases.
  • only contains "6 The miRNAs with a specific base of -11 dT" sequences can be complementary to the S-Oligo (dT) primers, so the S-Oligo (dT) method can distinguish between mature miRNAs and pri-miRNAs and pre-miRNAs.
  • the minimum template amount for amplification of miRNA by S-Oligo (dT) method is l pg total RNA; the linear range of amplification is broad, covering 6 orders of magnitude (l pg -100 ng total RNA), and the correlation coefficient R value is higher than 0.99.
  • the sensitivity of the S-Oligo (dT) method is 6-10 times higher than that of the poly(A) polymeric alcohol tailing method.
  • the S-Oligo (dT) method can simultaneously synthesize the reverse transcript primers of different miRNAs and synthesize the cDNA synthesis of multiple miRNAs in the process of cDNA synthesis, which not only can effectively reduce the error caused by the sample loading, but also facilitate the high Flux analysis.
  • the S-Oligo (dT) method uses a universal probe, which greatly reduces the cost of testing and is therefore more suitable for promotion.
  • Example 1 Quantitative detection of hsa-miR-21 miRNA in Hda cells of female cervical cancer
  • RNAiso plus (TaKaRa), PolyA Polymerase (EPICENTRE), PrimeScript RT reagent Kit (TaKaRa), SYBR Premix Ex Taq (Perfect Real Time) (TaKaRa), Premix Ex Taq (Perfect Real Time) (TaKaRa).
  • RNA extraction was performed using RNA iso plus reagent. Total RNA extracted from Hela cells was measured for OD260/280 and RNA concentrations using a spectrophotometer, and RNA integrity was analyzed by agarose gel electrophoresis.
  • RNA-PolyA (1 pg-100 ng / ⁇ ) ( ⁇ ) 2
  • Universal probes can be selected from any of TaqMan UnivP- 1, 2, 3, TaqMan UnivP-1, 2 is a LNA-modified probe primer. In this case, TaqMan UnivP-3 was selected.
  • PCR samples were placed on an ABI PRISM 7300 PCR machine and subjected to real-time PCR as follows: pre-denaturation at 95 °C for 30 seconds; denaturation at 95 °C for 5 seconds, extension at 60 °C for 31 seconds; 40 cycles.
  • This protocol uses a universal probe for the detection of PCR products.
  • the sequence is derived from the 17-base universal probe sequence on the S-Oligo (dT) primer.
  • the SYBR fluorescent dye method can also be used to detect the PCR product, ie SYBR. Green I fluorescent dye replaces the universal probe (TaqMan universal probe).
  • melting curve analysis can be performed to identify the specificity of the PCR product.
  • the hsa-miR-21 miRNA was quantitatively detected by S-Oligo (dT) method, and the S-Oligo (dT) method is shown in Figure 1.
  • the results showed that the hsa-miR-21 miRNA was well amplified in the range of 1 pg-100 ng of total RNA, as shown in Figure 2; the linear relationship of the standard curve was good, as shown in Figure 3.
  • This example also compares the sensitivity of different real-time RT-PCR methods including S-Oligo (dT), poly(A) polymeric alcohol tailing and stem loop primers to amplify hsa-miR-21.
  • Real-time RT-PCR was performed using the above three methods using 2 ng of total RNA as a template. The results are shown in Figure 4, S-dT The curve represents the result curve of the S-Oligo (dT) method, PolyA represents the result curve of the poly(A) polymerase tailing method, and SL represents the result curve of the stem loop primer method.
  • the average Ct value of the S-Oligo (dT) method is 21.8
  • the average Ct value of the poly(A) polymeric alcohol tailing method is 24.2
  • the average Ct value of the stem-loop primer method It is 26.1, so the sensitivity of the S-Oligo (dT) method is significantly higher than that of the poly(A) polymerase tailing method and the stem-loop primer method.

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Description

检测 miRNA的方法及引物及其应用
技术领域
本发明涉及生物医学领域，特别涉及一种检测 miRNA的方法及引物及 其在肿瘤等重大疾病早期诊断和预后中的应用。 背景技术
microRNA (miRNA) 是一类长约 22 个核苷酸的非编码单链小分子 RNA,广泛存在于动物，植物，线虫等真核生物中。 miRNA通过与靶 mRNA 3，非编码区 (3，UTR)的特异性结合， 降解靶 mRNA或阻遏其转录后翻译， 最 终导致靶基因的蛋白质合成减少。 miRNAs 参与了生命活动各个层次的调 节， 包括胚胎发育， 器官形成， 细胞增殖， 细胞凋亡， 细胞应激应答， 干 细胞分化， 内分泌调控， 免疫调节和疾病的发生与发展。 大量的研究结果 表明， 恶性肿瘤等重大疾病的发生和发展与人体内 miRNAs 的异常表达有 密切关系， 这就意味着 miRNAs 将有巨大的潜力成为新的生物标志物用于 癌症等重大疾病的早期诊断并可作为新的基因药物作用靶点。 显然， 要想 把 miRNA用于癌症等疾病的早期诊断， 需要一种能够快速、 灵敏、 特异地 定量检测 miRNAs表达的方法。
成熟 miRNA由于片段小， 没有 poly(A)尾巴， 不同 miRNA之间序列差 异小 （有些只有 1个核苷酸的差别）， 并且在细胞中的表达水平普遍较低， 这些都给 miRNA的定量检测工作带来了困难和挑战。 尽管如此， 科学家还 是研究出了多种 miRNA检测方法， 大体可以归为两类： 一类是不需要样本 扩增的探针直接杂交法； 另一类是基于 PCR扩增的 miRNA检测方法。
基于探针杂交技术的 miRNA检测方法是一种直接检测法，不需要对样 本 RNA进行预扩增， 主要有如下几种： Northern blot技术、 生物芯片技术 和基于微球的流式细胞术。这些技术的基本原理是将探针与 RNA样品进行 杂交， 然后进行信号检测。 Northern blot技术作为检测 RNA的经典方法， 常用来评价其它方法的可靠性； 但这种技术对样品要求量大， 操作相对费 时费力， 不适合高通量分析。 生物芯片技术能实现 miRNA的高通量分析， 即在一块芯片上同时检测多个 miRNA;但缺点是结果准确性低，重复性差， 实验价格昂贵。 基于微球的流式细胞术技术将探针固定于微球上并置于液 相中， 更有利于捕获 miRNA序列， 因此提高了准确性； 但该技术须使用特 殊的流式细胞仪和微球， 实验成本高， 同样不利于推广。
相比探针直接杂交法而言， 基于 real-time RT-PCR技术检测 miRNA的 表达是当前 miRNA研究中最为常用的技术手段之一，几乎所有依据高通量 芯片技术筛选出差异表达的 miRNA都需要用 real-time RT-PCR做进一步的 鉴定。基于 real-time RT-PCR的 miRNA检测方法， 主要包括 poly(A)聚合酶 加尾法（Shi R, Chiang V. Biotechnique. 2005, 39(4): 519-525 )和茎环引物
( Stem loop )法（ Chen C, Ridzon D. Nucleic Acids Res. 2005, 33(20): 1-9 )两 种。 poly(A)聚合酶加尾法是先用 poly(A)聚合酶使 miRNA的 3，端带上一段 poly(A)尾巴， 然后用 5，端含有接头序列的 Oligo (dT)引物进行反转录，使第 一链 cDNA加上一段接头， 为随后的 PCR扩增提供反向通用引物序列， 然 后再利用一条 miRNA序列特异的正向引物就可实现 PCR扩增。 该方法的 优点是筒便、快速，反转录引物对 miRNA具有通用性， 因此检测成本较低； 但通用反转录引物的使用降低了检测的特异性和灵敏性。 茎环引物法使用
3，端有 6个碱基与 miRNA互补配对的引物来进行 miRNA的反转录， 同时 反转录引物的 5，端含有一个茎环结构， 能增强 miRNA和 DNA异质双链的 亲合力， 并防止引物与 pri-或 pre-miRNA退火， 因此一般来讲茎环引物法 的特异性比 poly(A)聚合醇加尾法要好;但由于该法使用的是序列特异探针， 对于高通量的 miRNA分析来说过于昂贵。
因此， 现有技术还有待于改进和发展。
发明内容
鉴于上述现有技术的不足，本发明的目的在于提供一种检测 miRNA的 方法及引物及其应用， 旨在解决现有技术中所存在的问题。
本发明的技术方案如下：
一种用于定量检测 miRNA的 S-Oligo(dT)引物，其中，所述 S-Oligo(dT) 引物从 5，端开始由 PCR通用引物序列、 通用探针序列、 Oligo(dT)及与目的 miRNA分子的 3，端核苷酸互补配对的特异性序列 4部分组成。
所述的用于定量检测 miRNA的 S-Oligo(dT)引物，其中，所述 S-Oligo(dT) 引物从 5，端开始依次是 14-20个碱基的 PCR通用引物序列， 14~20个碱基 的通用探针序列， 8~30个 dT及与目的 miRNA分子的 3，端 3~8个核苷酸互 补配对的特异性序列。
所述的用于定量检测 miRNA的 S-Oligo(dT)引物，其中，所述引物从 5， 端开始依次是 16个碱基的 PCR通用引物序列， 17个碱基的通用探针序列， 11个 dT及与目的 miRNA分子的 3，端 6个核苷酸互补配对的特异性序列。
一种使用上述的用于定量检测 miRNA的引物定量检测特定 miRNA的 方法， 其中， 包括以下步骤：
S100、 S-Oligo(dT)引物和 miRNA 的特异上游引物的设计： 根据目的 miRNA的序列信息， 设计 S-Oligo(dT)引物和 miRNA的特异上游引物；
S200、 总 RNA提取： 将细胞裂解， 提取总 RNA, 并鉴定 RNA纯度和 完整性、 测定 RNA的浓度；
S300、 RNA加尾： 用 PolyA polymerase对总 RNA进行加尾， 得到 RNA-PolyA;
S400、 第一链 cDNA的合成： 以 RNA-PolyA为模板， 用 S-Oligo(dT) 引物进行 miRNA的反转录；
S500、 miRNA的 real-time PCR定量检测： 以 miRNA的第一链 cDNA 为模板， 用 miRNA特异上游引物和下游通用引物进行 real-time PCR定量 检测， 然后对 PCR结果进行数据分析。
所述的定量检测 miRNA的方法， 其中， 所述 miRNA特异上游引物是 不含 3，端 3~8个碱基的 miRNA特异序列， 所述 miRNA的下游通用引物来 自于 S-Oligo(dT)引物的 14~20个碱基的通用引物序列。 所述的定量检测 miRNA的方法，其中，步骤 S500中进行 real-time PCR 定量检测采用的是探针法或 SYBR荧光染料法； 所述探针法所用探针为通 用探针，其序列来自于 S-Oligo(dT)引物上的 14~20个碱基的通用探针序歹 ll。
一种使用上述的用于定量检测 miRNA的引物定量检测不同的 miRNA 的方法， 其中， 包括以下步骤：
S100、 S-Oligo(dT)引物和 miRNA的特异上游引物的设计： 根据不同的 目的 miRNA的序列信息， 设计不同的 S-Oligo(dT)引物和 miRNA的特异上 游引物；
S200、 总 RNA提取： 将细胞裂解， 提取总 RNA, 并鉴定 RN A纯度和 完整性鉴定和测定 RNA的浓度；
S300、 RNA加尾： 用 PolyA polymerase对总 RNA进行加尾， 得到 RNA-PolyA;
S400、 第一链 cDNA 的合成： 以 RNA-PolyA 为模板， 将不同的 S-Oligo(dT)引物组合起来进行 miRNA的反转录；
S500、 miRNA的 real-time PCR定量检测： 以 miRNA的第一链 cDNA 为模板， 用各个 miRNA特异上游引物和下游通用引物进行 real-time PCR 定量检测， 然后对 PCR结果进行数据分析。
所述的定量检测 miRNA的方法， 其中， 所述 miRNA特异上游引物是 不含 3，端 3~8个碱基的 miRNA特异序列， 所述 miRNA的下游通用引物来 自于 S-Oligo(dT)引物的 14~20个碱基的通用引物序列。
所述的定量检测 miRNA的方法，其中，步骤 S500中进行 real-time PCR 定量检测采用的是探针法或 SYBR荧光染料法； 所述探针法所用探针为通 用探针，其序列来自于 S-Oligo(dT)引物上的 14~20个碱基的通用探针序歹 ll。
上述的定量检测 miRNA 的方法的应用， 其中， 将所述的定量检测 miRNA的方法应用在肿瘤等重大疾病的早期诊断和预后中。
有益效果： 本方法的特点是灵敏性和特异性显著高于传统方法， 可高 通量分析， 操作筒单、 快速， 成本低， 可广泛应用于肿瘤等重大疾病的早 期诊断和预后。 附图说明
图 1为本发明用于 miRNA定量检测的 S-Oligo(dT)法示意图。
图 2 为本发明 S-Oligo(dT)法对女性宫颈癌 Hda细胞 hsa-miR-21 miRNA的扩增曲线图。
图 3 为本发明 S-Oligo(dT)法对女性宫颈癌 Hda细胞 hsa-miR-21 miRNA的扩增的标准曲线图。
图 4 为本发明实施例中不同 real-time RT-PCR法扩增 hsa-miR-21的灵 敏性比较曲线图。
具体实施方式 本发明提供一种检测 miRNA的方法及引物及其应用，为使本发明的目 的、 技术方案及效果更加清楚、 明确， 以下对本发明进一步详细说明。 应 当理解， 此处所描述的具体实施例仅仅用以解释本发明， 并不用于限定本 发明。
本发明所提供的用于检测 miRNA 的 miRNA 反转录引物—— S-Oligo(dT)引物，从 5，端开始由 PCR通用引物序列（Universal primer)、通用 探针序列（Universal probe)、01igo(dT)及与目的 miRNA分子的 3，端核苷酸互 补配对的特异性序列（specific bases)4部分组成。
所述用于定量检测 miRNA 的 S-Oligo(dT)引物， 从 5，端开始依次是 14-20个碱基的 PCR通用引物序列， 14~20个碱基的通用探针序列， 8~30 个 dT及与目的 miRNA分子的 3，端 3~8个核苷酸互补配对的特异性序列。
本发明还提供 miRNA 定量检测的一种新方法—— S-Oligo(dT)法。 S-Oligo(dT)法运用 S-Oligo(dT)引物进行特定 miRNA的反转录，或者将不同 S-Oligo(dT)引物组合起来对不同的 miRNA样品同时进行反转录， 然后用各 个 miRNA的特异上游引物和通用下游引物对相应的 miRNA进行 real-time PCR定量检测。 miRNA特异上游引物是不含 3，端 3~8个碱基的 miRNA特 异序列， miRNA的下游通用引物来自于 S-Oligo(dT)引物的 14~20个碱基的 通用引物序列。 PCR产物的检测既可用荧光染料法也可用探针法。 探针法 使用通用探针， 其序列来自于 S-Oligo(dT)引物上 14~20个碱基的通用探针 序列。
本发明实施例中所提供的 S-Oligo(dT)引物， 其组成为： 从引物 5，端开 始依次是 16个碱基的 PCR通用引物序列， 17个碱基的通用探针序列， 11 个 dT (Oligo(dT))及与目的 miRNA分子的 3，端 6个核苷酸互补配对的特异 性序列。 miRNA特异上游引物是不含 3，端 6个碱基的 miRNA特异序列， miRNA的下游通用引物来自于 S-Oligo(dT)引物的 16个碱基的通用引物序 列。 所使用的通用探针， 其序列来自于 S-Oligo(dT)引物上 17个碱基的通用 探针序列。
本发明所提供的 miRNA定量检测方法—— S-Oligo(dT)法可以应用在肿 瘤等重大疾病的早期诊断和预测。
本发明 miRNA定量检测方法的具体步骤为：
S100、 S-Oligo(dT)引物和 miRNA 的特异上游引物的设计： 根据目的 miRNA的序列信息， 设计 S-Oligo(dT)引物和 miRNA的特异上游引物；
S200、 总 RNA提取： 将细胞裂解， 提取总 RNA, 并鉴定 RN A纯度和 完整性、 测定 RNA的浓度；
S300、 RNA加尾： 用 PolyA polymerase对总 RNA进行加尾， 得到 RNA-PolyA;
S400、 第一链 cDNA的合成： 以 RNA-PolyA为模板， 用 S-Oligo(dT) 引物进行 miRNA的反转录；
S500、 miRNA的 real-time PCR定量检测： 以 miRNA的第一链 cDNA 为模板， 用 miRNA特异上游引物和下游通用引物进行 real-time PCR定量 检测， 然后对 PCR结果进行数据分析。
使用本发明的检测 miRNA的方法， 具有以下几大优点：
1. S-Oligo(dT)法检测 miRNA的特异性显著提高：
S-Oligo(dT)引物与靶 miRNA互补的碱基数为 17个碱基（ 6个特异碱基 和 11个 dT ),在与靶 miRNA锚定力度上明显优于 poly(A)聚合醇加尾法（只 有 Oligo(dT) )和茎环引物法（只有 6个特异碱基）。 同时， 只有包含 " 6个 特异碱基 -11个 dT"序列的 miRNA才能与 S-Oligo(dT)引物互补配对， 因此 S-Oligo(dT)法能很好地区分成熟 miRNA与 pri-miRNA和 pre-miRNA。
2. S-Oligo(dT)法检测 miRNA的灵敏性高：
S-Oligo(dT)法扩增 miRNA的最低模板量为 l pg 总 RNA; 扩增线性范 围宽， 能覆盖 6个数量级（l pg -100 ng 总 RNA ),相关系数 R值高于 0.99。 S-Oligo(dT)法与茎环引物法和 poly(A)聚合醇加尾法相比灵敏性能提高 6-10 倍。
3.操作筒单， 能进行高通量分析：
S-Oligo(dT)法在 cDNA合成过程中，能同时将不同 miRNA的反转录引 物混合后进行多个 miRNA的 cDNA合成，这样不仅能有效降低因加样所导 致的误差， 而且有利于高通量分析。
4.检测成本低：
S-Oligo(dT)法使用通用探针， 大大降低了检测成本， 因此更加适合推广。 实施例 1 女性宫颈癌 Hda细胞 hsa-miR-21 miRNA的定量检测
(一 )材料
1. 材料：
体外培养的女性宫颈癌 Hda细胞。
2. 试剂：
RNAiso plus (TaKaRa), PolyA Polymerase (EPICENTRE), PrimeScript RT reagent Kit (TaKaRa), SYBR Premix Ex Taq (Perfect Real Time) (TaKaRa) , Premix Ex Taq (Perfect Real Time) (TaKaRa)。
3. 引物和探针:
？ ]物 /探针名称 ]物 /探针序列 im
miR-21U 5 ' -GCCCGCTAGCTTATCAGACTGATG-3 ' hsa-miR-21上游引物
5 ' -GTGC AGGGTCCGAGGTCAGAGCCAC hsa-miR-21反转录引物 miR21-SRTP
CTGGGCAATTTTTTTTTTTTCAACA-3 '
3UnivP 5， -GTGCAGGGTCCGAGGT-3 ' miRNA通用下游引物
TaqMan UnivP- 1 5， - TTGCCCAGG -3' miRNA通用探针 1
TaqMan UnivP-2 5 ' - TGGCTCTG -3' miRNA通用探针 2
TaqMan UnivP-3 5 ' - TTGCCCAGGTGGCTCTG -3' miRNA通用探针 3 4. 仪器：
GeneQuant pro RNA/DNA 定量分析仪， ABI PCR仪（2720 ) , ABI real-time PCR仪（ PRISM 7300 )。
(二） 方法
1. RNA提取
将体外培养的 Hela细胞， 用 RNAiso plus试剂进行 RNA提取。 提取的 Hela细胞总 RNA用分光光度计测定 OD260/280及 RNA浓度， 用琼脂糖凝 胶电泳分析 RNA的完整性。
2. RNA加尾反应
用 PolyA Polymerase对总 RNA进行力口尾反应， 力口样如下：
RNA (0. 5μ /μ1) 2
10 x Reaction Buffer (μΐ) 1
ΙΟ ιηΜ ΑΤΡ (μΙ) 1
Poly(A) Polymerase (4υ/μ1) 0.5
RNase-Free Water (μΐ)
Total volume (μΐ) 10
加完样后， 37。C保温 30min, 冰上保存。
3. 第一链 cDNA的合成
用 PrimeScript RT reagent Kit对 miRNA进行反转录反应， 力口样: ¾口下：
RNA-PolyA (1 pg-100 ng /μΐ) (μΐ) 2
0.5 μΜ miR21-SRTP primer (μΐ) 1
H2Q (μΐ) 3
Total vol (μΐ) 6
加完样后， 65。C保温 5 min, 迅速置于冰上， 静置 2min。 加入如下试 剂：
5 X PrimeScript Buffer (μΐ) 2
PrimeScript RT Enzyme (μΐ) 0.5
H2Q (μΐ) L5
Total vol (μΐ) 4
然后， 42 °C保温 15 min, 85 °C保温 5 min, 冰上放置； 再加入纯水 34 μΐ, 混匀。
4. Hsa-miR-21 miRNA的 real-time PCR定量检测
用 Premix Ex Taq™ (Perfect Real Time)进行 hsa-miR-21 miRNA 的 real-time PCR定量检测， 力 P样如下：
Premix Ex Taq (2 x ) (μΐ) 10
10 μΜ miR-21U (μΐ) 0.4
10 μΜ 3UnivP (μΐ) 0.4
6 μΜ TaqMan UnivP * 0.4
hsa-miR-21 cDNA (μΐ) 8,8
Total vol (μΐ) 20
* 通用探针可以选择 TaqMan UnivP- 1, 2, 3三种中任意一种， TaqMan UnivP- 1 , 2为 LNA修饰的探针引物。 本例选用 TaqMan UnivP-3。
将 PCR样品置于 ABI PRISM 7300 PCR仪，按如下条件进行 real-time PCR: 95°C预变性 30秒； 95°C变性 5秒， 60°C延伸 31秒； 40个循环。
本方案采用通用探针进行 PCR产物的检测， 其序列来自 S-Oligo(dT) 引物上 17个碱基的通用探针序列； 也可以使用 SYBR荧光染料法对进行 PCR 产物的检测， 即用 SYBR Green I 荧光染料代替通用探针 (TaqMan universal probe)。 使用 SYBR Green I荧光染料时， 可进行熔解曲线分析， 以鉴定 PCR产物的特异性。
(三） 结果
1. S-Oligo(dT)法对 hsa-miR-21 miRNA进行定量检测
从女性宫颈癌 Hela细胞提取的总 RNA经琼脂糖凝胶电泳鉴定表明完 整性良好； OD26。/28。 = 1.9 , 表明 RNA纯度较高。 于是用 S-Oligo(dT)法对 hsa-miR-21 miRNA进行定量检测， S-Oligo(dT)法示意图如图 1所示。 结果 表明， hsa-miR-21 miRNA在模板为 1 pg- 100 ng的总 RNA范围内都得到了 较好的扩增， 如图 2所示； 标准曲线的线性关系良好， 如图 3所示。
2. 不同 real-time RT-PCR方法的灵敏性比较
本实施例还比较了不同 real-time RT-PCR方法（包括 S-Oligo(dT)法、 poly(A)聚合醇加尾法和茎环引物法 )扩增 hsa-miR-21的灵敏性。 以 2 ng总 RNA为模板，用上述三种方法进行 real-time RT-PCR检测，结果见图 4, S-dT 曲线表示 S-Oligo(dT)法的结果曲线， PolyA表示 poly(A)聚合酶加尾法的结 果曲线， SL表示茎环引物法的结果曲线。从图 4荧光强度与 Ct值关系曲线 看， S-Oligo(dT)法的平均 Ct值为 21.8, poly(A)聚合醇加尾法的平均 Ct值 为 24.2, 茎环引物法的平均 Ct值为 26.1 , 因此 S-Oligo(dT)法的灵敏性明显 高于 poly(A)聚合酶加尾法和茎环引物法。
应当理解的是， 本发明的应用不限于上述的举例， 对本领域普通技术 人员来说， 可以根据上述说明加以改进或变换， 所有这些改进和变换都应 属于本发明所附权利要求的保护范围。

Claims

权 利 要 求 书
1、 一种用于定量检测 miRNA的 S-Oligo(dT)引物， 其特征在于， 所述 S-Oligo(dT)引物从 5，端开始由 PCR通用引物序列、通用探针序列、 Oligo(dT) 及与目的 miRNA分子的 3，端核苷酸互补配对的特异性序列 4部分组成。
2、 根据权利要求 1所述的用于定量检测 miRNA的 S-Oligo(dT)引物， 其特征在于， 所述 S-Oligo(dT)引物从 5，端开始依次是 14-20个碱基的 PCR 通用引物序列， 14~20个碱基的通用探针序列， 8~30个 dT及与目的 miRNA 分子的 3，端 3~8个核苷酸互补配对的特异性序列。
3、 根据权利要求 1所述的用于定量检测 miRNA的 S-Oligo(dT)引物， 其特征在于， 所述引物从 5，端开始依次是 16个碱基的 PCR通用引物序列， 17个碱基的通用探针序列， 11个 dT及与目的 miRNA分子的 3，端 6个核苷 酸互补配对的特异性序列。
4、 一种使用权利要求 1所述的用于定量检测 miRNA的引物定量检测 特定 miRNA的方法， 其特征在于， 包括以下步骤：
S100、 S-Oligo(dT)引物和 miRNA 的特异上游引物的设计： 根据目的 miRNA的序列信息， 设计 S-Oligo(dT)引物和 miRNA的特异上游引物；
S200、 总 RNA提取： 将细胞裂解， 提取总 RNA, 并鉴定 RN A纯度和 完整性、 测定 RNA的浓度；
S300、 RNA加尾： 用 PolyA polymerase对总 RNA进行加尾， 得到 RNA-PolyA;
S400、 第一链 cDNA的合成： 以 RNA-PolyA为模板， 用 S-Oligo(dT) 引物进行 miRNA的反转录；
S500、 miRNA的 real-time PCR定量检测： 以 miRNA的第一链 cDNA 为模板， 用 miRNA特异上游引物和下游通用引物进行 real-time PCR定量 检测， 然后对 PCR结果进行数据分析。
5、 根据权利要求 4所述的定量检测 miRNA的方法， 其特征在于， 所 述 miRNA特异上游引物是不含 3，端 3~8个碱基的 miRNA特异序列， 所述 miRNA的下游通用引物来自于 S-Oligo(dT)引物的 14~20个碱基的通用引物 序列。
6、 根据权利要求 4所述的定量检测 miRNA的方法， 其特征在于， 步 骤 S500中进行 real-time PCR定量检测采用的是探针法或 SYBR荧光染料 法； 所述探针法所用探针为通用探针， 其序列来自于 S-Oligo(dT)引物上的 14-20个碱基的通用探针序列。
7、 一种使用权利要求 1所述的用于定量检测 miRNA的引物定量检测 不同的 miRNA的方法， 其特征在于， 包括以下步骤：
S100、 S-Oligo(dT)引物和 miRNA的特异上游引物的设计： 根据不同的 目的 miRNA的序列信息， 设计不同的 S-Oligo(dT)引物和 miRNA的特异上 游引物；
S200、 总 RNA提取： 将细胞裂解， 提取总 RNA, 并鉴定 RNA纯度和 完整性鉴定和测定 RNA的浓度；
S300、 RNA加尾： 用 PolyA polymerase对总 RNA进行加尾， 得到 RNA-PolyA;
S400、 第一链 cDNA 的合成： 以 RNA-PolyA 为模板， 将不同的 S-Oligo(dT)引物组合起来进行 miRNA的反转录；
S500、 miRNA的 real-time PCR定量检测： 以 miRNA的第一链 cDNA 为模板， 用各个 miRNA特异上游引物和下游通用引物进行 real-time PCR 定量检测， 然后对 PCR结果进行数据分析。
8、 根据权利要求 7所述的定量检测 miRNA的方法， 其特征在于， 所 述 miRNA特异上游引物是不含 3，端 3~8个碱基的 miRNA特异序列， 所述 miRNA的下游通用引物来自于 S-Oligo(dT)引物的 14~20个碱基的通用引物 序列。
9、 根据权利要求 7所述的定量检测 miRNA的方法， 其特征在于， 步 骤 S500中进行 real-time PCR定量检测采用的是探针法或 SYBR荧光染料 法； 所述探针法所用探针为通用探针， 其序列来自于 S-Oligo(dT)引物上的 14-20个碱基的通用探针序列。
10、 一种权利要求 4或 7所述的定量检测 miRNA的方法的应用， 其特 征在于，将所述的定量检测 miRNA的方法应用在肿瘤等重大疾病的早期诊 断和预后中。
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