技术领域technical field
本发明属于生物技术和肿瘤学领域，尤其是涉及膀胱癌组织中piRNA生物标志物及其表达量的检测方法。The invention belongs to the fields of biotechnology and oncology, and in particular relates to a method for detecting piRNA biomarkers and their expression levels in bladder cancer tissues.
背景技术：Background technique:
膀胱癌是泌尿系统最常见的恶性肿瘤之一，全球，膀胱癌在男性肿瘤中位列第七，在女性中位列第十七。在我国泌尿系统肿瘤中，膀胱癌无论是发病率还是死亡率均居首位并且发病率呈逐年升高趋势，大部分患者年龄介于50-60岁之间。膀胱癌90%以上为移行上皮细胞癌，按病理分期可分为浅表性膀胱癌(早期）和肌层浸润性膀胱癌（进展期）。膀胱癌的发生发展，可严重影响患者的生存及生活质量，因此寻找敏感特异的可用于早期诊断的生物学标志物，以期达到利用其开展预防、筛检及诊断，从而改善膀胱癌患者的生存及生活质量。Bladder cancer is one of the most common malignant tumors of the urinary system. Globally, bladder cancer ranks seventh among male tumors and seventeenth among females. Among urinary system tumors in my country, bladder cancer ranks first in terms of morbidity and mortality, and the incidence rate is increasing year by year. Most patients are between 50 and 60 years old. More than 90% of bladder cancer is transitional epithelial cell carcinoma, which can be divided into superficial bladder cancer (early stage) and muscle-invasive bladder cancer (advanced stage) according to pathological stage. The occurrence and development of bladder cancer can seriously affect the survival and quality of life of patients. Therefore, we are looking for sensitive and specific biomarkers that can be used for early diagnosis, in order to use them to carry out prevention, screening and diagnosis, so as to improve the survival of patients with bladder cancer and quality of life.
RNA的研究近年来备受科学界的广泛关注，尤其是一些非编码的小RNA（non-codingRNA，ncRNA）分子，这些ncRNA分子可以调节细胞生长和发育的重要生理过程。ncRNA调控细胞的生命过程包括转录水平和转录后水平的基因沉默。一般小ncRNA可以分为三类：siRNAs，microRNAs（miRNAs）和PIWI-interactingRNAs（PIWI蛋白相关非编码RNAs，简称piRNAs）[2]。其中piRNA的研究比较少，piRNA潜在的功能和生物起源与miRNA和siRNA存在明显不同。piRNA长度约26nt–31nt，可与PIWI蛋白结合形成piRNA复合物（piRC）而发挥其生物学功能。不同于miRNA，piRNA不是由含茎环结构的前体剪切产生。piRNA序列的基因组分布表明，piRNA通常成簇出现，并且有很强的正义链或反义链专一性。In recent years, the research on RNA has attracted extensive attention from the scientific community, especially some small non-coding RNA (non-coding RNA, ncRNA) molecules, which can regulate important physiological processes of cell growth and development. ncRNAs regulate the life processes of cells, including gene silencing at the transcriptional and post-transcriptional levels. Generally, small ncRNAs can be divided into three categories: siRNAs, microRNAs (miRNAs) and PIWI-interacting RNAs (PIWI protein-related non-coding RNAs, piRNAs for short)[2] . Among them, the research on piRNA is less, and the potential function and biological origin of piRNA are obviously different from those of miRNA and siRNA. The length of piRNA is about 26nt–31nt, and it can combine with PIWI protein to form piRNA complex (piRC) to exert its biological function. Unlike miRNAs, piRNAs are not produced by cleavage of precursors containing stem-loop structures. The genomic distribution of piRNA sequences shows that piRNAs usually appear in clusters and have strong sense-strand or antisense-strand specificity.
对于piRNA的产生机制，目前尚无直接的生物化学证据。但是一个基于生物信息学分析提出了“乒乓模型”假说。该模型认为：反义链的piRNA前体经过某些未知核酸酶的加工，生成了5’端具有U（尿嘧啶）偏爱性的初级piRNA，这些piRNA与Aub或Piwi蛋白结合形成piRC，piRC在piRNA的引导下，通过碱基互补配对的方式识别并结合正义链的piRNA前体，然后将正义链piRNA前体进行剪切切割，产生次级piRNA的5’端，而后该次级piRNA被某种核酸酶切割形成3’端，最后生成成熟的正义链piRNA。正义链piRNA与Ago3（PIWI）结合形成piRC，通过类似的方式识别并切割反义链的piRNA前体。这样就形成了一个piRNA生物发生循环，正义链与反义链的相互识别，通过piRC的剪切切割，循环产生piRNA。目前该“乒乓模型”假说，得到了许多学者的广泛认同。There is no direct biochemical evidence for the mechanism of piRNA production. But a "ping-pong model" hypothesis was proposed based on bioinformatics analysis. The model believes that the piRNA precursors of the antisense strand are processed by some unknown nucleases to generate primary piRNAs with a U (uracil) preference at the 5' end, and these piRNAs combine with Aub or Piwi proteins to form piRC, and piRC is in the Under the guidance of piRNA, it recognizes and binds the piRNA precursor of the sense strand through complementary base pairing, and then cuts the sense strand piRNA precursor to generate the 5' end of the secondary piRNA, and then the secondary piRNA is captured by a certain A nuclease cuts to form the 3' end, and finally generates a mature sense-strand piRNA. The sense strand piRNA binds to Ago3 (PIWI) to form piRC, which recognizes and cleaves the antisense strand piRNA precursor in a similar way. In this way, a piRNA biogenesis cycle is formed, the mutual recognition of the sense strand and the antisense strand, and the cleavage and cleavage of piRC produce piRNA in a cycle. At present, the "ping-pong model" hypothesis has been widely recognized by many scholars.
值得注意的是，在2006年7月的Nature杂志上面，两个独立的课题组同期发表了关于piRNA的文章，Aravin等运用Northernblot的方法证实只有生殖细胞中才存在piRNA，同时Girard等也证明了这种存在形式。近来，也有不少研究报道了piRNA与人类肿瘤之间的相关性，但是机制方面的研究较少。如Lu等采用Solexa测序的方法，发现piRNA在人宫颈癌HeLa细胞中存在，Chen等也发现piRNA存在于人的多种肿瘤细胞中。另外Chen等在piRNA的研究中发现，piRNA-651与胃癌的临床分期显著相关，同时观察到其在胃癌、结肠癌、肺癌以及乳腺癌组织中的表达水平显著高于其对应的癌旁组织，提示该piRNA可以作为肿瘤诊断潜在的生物标志物。It is worth noting that in the July 2006 issue of Nature, two independent research groups published articles on piRNA at the same time. Aravin et al. used the method of Northern blot to confirm that piRNA exists only in germ cells. At the same time, Girard et al. also proved that this form of existence. Recently, many studies have reported the correlation between piRNA and human tumors, but there are few studies on the mechanism. For example, Lu et al. used Solexa sequencing to find that piRNA exists in human cervical cancer HeLa cells, and Chen et al. also found that piRNA exists in various human tumor cells. In addition, in the study of piRNA, Chen et al. found that piRNA-651 was significantly correlated with the clinical stage of gastric cancer, and observed that its expression level in gastric cancer, colon cancer, lung cancer and breast cancer tissues was significantly higher than that in the corresponding paracancerous tissues. It suggested that the piRNA could be used as a potential biomarker for tumor diagnosis.
目前对于膀胱组织中piRNA的提取及检测尚缺乏，另现有技术中均没有涉及到膀胱癌组织及其对应癌旁正常组织中与哪些piRNA相关。At present, the extraction and detection of piRNA in bladder tissue is still lacking, and none of the prior art involves which piRNA is related to bladder cancer tissue and its corresponding adjacent normal tissue.
发明内容Contents of the invention
解决的技术问题：本发明的目的在于克服现有技术中的不足，提供一种用于诊断膀胱癌的piRNA生物标志物及其应用，建立膀胱癌患者组织piRNA的表达谱，揭示组织中piRNA对膀胱癌诊断的价值，为临床上膀胱癌早期发现提供基础。Technical problem to be solved: the purpose of the present invention is to overcome the deficiencies in the prior art, provide a kind of piRNA biomarker and its application for diagnosing bladder cancer, establish the expression profile of bladder cancer patient tissue piRNA, reveal the piRNA in tissue The value of bladder cancer diagnosis provides the basis for early detection of bladder cancer in clinic.
技术方案：一种与膀胱癌相关的piRNA生物标志物，所述生物标志物：piRNA共197个，其中：Technical solution: a piRNA biomarker related to bladder cancer, said biomarker: a total of 197 piRNAs, of which:
上调的106个，如SEQIDNo.1～106所示：piR-52681、piR-48765、piR-36984、piR-52373、piR-31112、piR-36360、piR-61291、piR-54826、piR-51747、piR-43148、piR-45726、piR-48368、piR-58119、piR-57089、piR-39017、piR-43887、piR-37525、piR-39614、piR-39018、piR-35831、piR-39592、piR-33050、piR-34998、piR-38355、piR-56914、piR-34920、piR-37523、piR-45582、piR-51256、piR-37524、piR-52680、piR-31111、piR-56252、piR-58255、piR-31101、piR-30597、piR-32079、piR-54098、piR-34984、piR-31108、piR-34000、piR-31961、piR-33650、piR-57293、piR-33470、piR-36475、piR-47132、piR-45029、piR-58050、piR-36707、piR-60668、piR-34756、piR-47161、piR-55637、piR-44190、piR-31013、piR-50437、piR-33543、piR-43217、piR-38376、piR-50657、piR-61404、piR-51364、piR-48118、piR-30327、piR-54136、piR-45459、piR-44085、piR-52697、piR-49813、piR-57447、piR-39232、piR-57132、piR-39763、piR-45051、piR-33043、piR-53177、piR-43105、piR-57140、piR-40110、piR-37972、piR-40982、piR-42947、piR-43876、piR-38354、piR-57141、piR-57567、piR-44956、piR-33368、piR-56037、piR-57143、piR-43107、piR-33733、piR-57142、piR-57139、piR-34534、piR-56276、piR-32683、piR-55475、piR-34532、piR-33730、piR-59160、piR-61324、piR-49600、piR-52323、piR-38240；Up-regulated 106, as shown in SEQIDNo.1-106: piR-52681, piR-48765, piR-36984, piR-52373, piR-31112, piR-36360, piR-61291, piR-54826, piR-51747, piR-43148, piR-45726, piR-48368, piR-58119, piR-57089, piR-39017, piR-43887, piR-37525, piR-39614, piR-39018, piR-35831, piR-39592, piR- 33050, piR-34998, piR-38355, piR-56914, piR-34920, piR-37523, piR-45582, piR-51256, piR-37524, piR-52680, piR-31111, piR-56252, piR-58255, piR-31101, piR-30597, piR-32079, piR-54098, piR-34984, piR-31108, piR-34000, piR-31961, piR-33650, piR-57293, piR-33470, piR-36475, piR- 47132, piR-45029, piR-58050, piR-36707, piR-60668, piR-34756, piR-47161, piR-55637, piR-44190, piR-31013, piR-50437, piR-33543, piR-43217, piR-38376, piR-50657, piR-61404, piR-51364, piR-48118, piR-30327, piR-54136, piR-45459, piR-44085, piR-52697, piR-49813, piR-57447, piR- 39232, piR-57132, piR-39763, piR-45051, piR-33043, piR-53177, piR-43105, piR-57140, piR-40110, piR-37972, piR-40982, piR-42947, piR-43876, piR-38354, piR-57141, piR-57567, piR-44956, piR-33368, piR-56037, piR-57143, piR-43107, piR-33733, piR-57142, piR-57139, piR-34534, piR- 56276, piR-3 2683, piR-55475, piR-34532, piR-33730, piR-59160, piR-61324, piR-49600, piR-52323, piR-38240;
下调的91个，如SEQIDNo.107～197所示，：piR-60152、piR-34373、piR-30504、piR-34374、piR-55023、piR-34375、piR-43452、piR-52155、piR-34380、piR-34379、piR-44265、piR-40154、piR-34420、piR-48578、piR-35688、piR-56066、piR-37243、piR-47995、piR-49208、piR-34377、piR-56061、piR-34376、piR-38145、piR-58986、piR-30506、piR-37988、piR-33851、piR-39786、piR-56470、piR-43801、piR-45852、piR-58572、piR-49842、piR-49039、piR-51517、piR-35536、piR-35924、piR-33814、piR-47258、piR-32538、piR-30436、piR-52398、piR-36439、piR-34177、piR-52399、piR-35359、piR-31167、piR-56951、piR-42797、piR-40163、piR-31440、piR-32499、piR-34422、piR-31133、piR-36614、piR-45571、piR-32406、piR-46447、piR-34348、piR-31252、piR-45112、piR-54110、piR-58451、piR-49939、piR-49552、piR-39004、piR-30769、piR-40571、piR-50830、piR-45655、piR-42221、piR-35166、piR-35167、piR-39572、piR-39516、piR-38856、piR-35717、piR-33417、piR-38060、piR-55800、piR-33401、piR-39593、piR-35762、piR-53013、piR-35899、piR-34726、piR-33466、piR-34194、piR-54213、piR-58463、piR-34366。91 down-regulated ones, as shown in SEQIDNo.107-197: piR-60152, piR-34373, piR-30504, piR-34374, piR-55023, piR-34375, piR-43452, piR-52155, piR-34380 , piR-34379, piR-44265, piR-40154, piR-34420, piR-48578, piR-35688, piR-56066, piR-37243, piR-47995, piR-49208, piR-34377, piR-56061, piR -34376, piR-38145, piR-58986, piR-30506, piR-37988, piR-33851, piR-39786, piR-56470, piR-43801, piR-45852, piR-58572, piR-49842, piR-49039 , piR-51517, piR-35536, piR-35924, piR-33814, piR-47258, piR-32538, piR-30436, piR-52398, piR-36439, piR-34177, piR-52399, piR-35359, piR -31167, piR-56951, piR-42797, piR-40163, piR-31440, piR-32499, piR-34422, piR-31133, piR-36614, piR-45571, piR-32406, piR-46447, piR-34348 , piR-31252, piR-45112, piR-54110, piR-58451, piR-49939, piR-49552, piR-39004, piR-30769, piR-40571, piR-50830, piR-45655, piR-42221, piR -35166, piR-35167, piR-39572, piR-39516, piR-38856, piR-35717, piR-33417, piR-38060, piR-55800, piR-33401, piR-39593, piR-35762, piR-53013 , piR-35899, piR-34726, piR-33466, piR-34194, piR-54213, piR-58463, piR-34366.
一种与膀胱癌相关的piRNA生物标志物，所述生物标志物为上调的piR-52681和下调的piR-60152。A piRNA biomarker associated with bladder cancer, the biomarkers are upregulated piR-52681 and downregulated piR-60152.
所述的piRNA生物标志物在制备膀胱癌检测试剂盒中的应用。Application of the piRNA biomarker in the preparation of a bladder cancer detection kit.
与膀胱癌相关的piRNA生物标志物的检测及表达谱建立方法，包括以下步骤：A method for detecting and establishing an expression profile of piRNA biomarkers related to bladder cancer, comprising the following steps:
（1）使用Trizol试剂（Invitrogen）从膀胱癌患者的癌组织及其对应的癌旁组织中提取总RNA，通过ArrayStar的piRNA芯片筛选出候选差异表达的piRNA；(1) Using Trizol reagent (Invitrogen) to extract total RNA from cancer tissues of bladder cancer patients and their corresponding paracancerous tissues, and screen candidate differentially expressed piRNAs by ArrayStar piRNA chips;
（2）RNA逆转，采用miScript逆转录试剂盒（Qiagen）对RNA提取液进行逆转录反应，获得cDNA溶液；(2) RNA reversal, using the miScript reverse transcription kit (Qiagen) to carry out reverse transcription reaction on the RNA extraction solution to obtain cDNA solution;
（3）PCR检测，运用miScriptSYBRGreenPCR检测试剂盒对步骤（2）中的cDNA溶液进行PCR扩增反应，进而完成对候选piRNA的验证，建立膀胱癌piRNA表达谱。(3) PCR detection, using the miScriptSYBRGreen PCR detection kit to perform PCR amplification reaction on the cDNA solution in step (2), and then complete the verification of candidate piRNAs and establish the expression profile of bladder cancer piRNAs.
与膀胱癌相关的piRNA生物标志物表达量的检测方法，采用SYBRqRT-PCR的方法检测膀胱组织中piRNA的表达量：The method for detecting the expression level of piRNA biomarkers related to bladder cancer, using the SYBRqRT-PCR method to detect the expression level of piRNA in bladder tissue:
（1）逆转录：采用miScript逆转录试剂盒（Qiagen）对提取出的总RNA进行逆转录反应，具体操作依据该试剂盒的说明书进行，首先在0.5mL离心管中加入5μLDEPC双蒸水、继而加入1μL逆转录混合试剂和4μLRT缓冲液，最后加入10μL0.1μg/μL的总RNA，总反应体系20μL，该方法的反应条件为：37℃反应1小时，95℃反应5分钟，得到cDNA溶液；(1) Reverse transcription: Use the miScript reverse transcription kit (Qiagen) to carry out reverse transcription reaction on the extracted total RNA. The specific operation is carried out according to the instructions of the kit. Add 1 μL reverse transcription mixed reagent and 4 μL RT buffer, and finally add 10 μL 0.1 μg/μL total RNA, the total reaction system is 20 μL, the reaction conditions of this method are: 37 ° C for 1 hour, 95 ° C for 5 minutes to obtain cDNA solution;
（2）piR-52681实时荧光定量PCR检测：采用miScriptSYBRGreenPCR检测试剂盒对步骤（1）中获得的cDNA溶液进行PCR扩增反应，扩增引物为piR-52681扩增上游引物和PCR检测的小RNA通用的下游引物，再用ABI7900HTReal-TimePCR仪检测，具体操作依据该试剂盒与荧光定量PCR仪说明书进行，反应体系包括：1μLpiR-52681扩增上游引物和1μLPCR检测的小RNA通用的下游引物，5μLSYBRGreenPCR反应缓冲液，2μLDEPC双蒸水以及1μL0.1μg/μLcDNA稀释液，该方法的反应条件：95℃预变性15分钟，94℃变性15秒，60℃退火30秒，70℃延伸30秒，94℃-70℃的阶段共40个循环；(2) Real-time fluorescent quantitative PCR detection of piR-52681: Use miScriptSYBRGreenPCR detection kit to perform PCR amplification reaction on the cDNA solution obtained in step (1), and the amplification primer is piR-52681 to amplify the upstream primer and the small RNA detected by PCR The general downstream primers are then detected by ABI7900HTReal-Time PCR instrument. The specific operation is carried out according to the kit and fluorescence quantitative PCR instrument instructions. The reaction system includes: 1 μL piR-52681 amplification upstream primer and 1 μL PCR detection small RNA general downstream primer, 5 μL SYBRGreenPCR Reaction buffer, 2 μL DEPC double-distilled water and 1 μL 0.1 μg/μL cDNA diluent, the reaction conditions of this method: 95°C pre-denaturation for 15 minutes, 94°C denaturation for 15 seconds, 60°C annealing for 30 seconds, 70°C extension for 30 seconds, 94°C A total of 40 cycles in the -70°C stage;
（3）piR-60152实时荧光定量PCR检测：与步骤（2）中的方法相同，所不同的只是扩增的上游引物由piR-52681特异性扩增上游引物代替piR-60152，检测得到膀胱癌组织及癌旁组织中piR-60152的表达水平；(3) piR-60152 real-time fluorescence quantitative PCR detection: the same method as in step (2), the difference is that the amplified upstream primer is specifically amplified by piR-52681 instead of piR-60152, and bladder cancer is detected The expression level of piR-60152 in tissues and adjacent tissues;
（4）U6实时荧光定量PCR检测：与步骤（2）中的方法相同，所不同的只是扩增的上游引物有piR-52681特异性扩增上游引物代替U6，检测得到膀胱癌组织及癌旁组织中U6的表达水平；在本方案中U6作为piR-52681与piR-60152的内参。(4) U6 real-time fluorescent quantitative PCR detection: the same method as in step (2), the difference is that the amplified upstream primer has piR-52681 specific amplification upstream primer instead of U6, and the bladder cancer tissue and adjacent tumors can be detected The expression level of U6 in tissues; in this protocol, U6 is used as an internal reference for piR-52681 and piR-60152.
有益效果：本发明首次发现piRNA在膀胱癌临床早期鉴别诊断中，可作为一种重要的生物学检测指标，为今后膀胱癌piRNA的研究提供了理论依据，并为分子水平诊断膀胱癌提供了新的思路，具有重大的理论意义和潜在的使用价值。Beneficial effects: the present invention finds for the first time that piRNA can be used as an important biological detection index in the clinical early differential diagnosis of bladder cancer, which provides a theoretical basis for the future research on bladder cancer piRNA, and provides a new method for the diagnosis of bladder cancer at the molecular level. The idea has great theoretical significance and potential use value.
附图说明Description of drawings
图1为实时荧光定量PCR检测piR-52681在膀胱癌组织及癌旁组织中的表达；Figure 1 shows the expression of piR-52681 detected by real-time fluorescent quantitative PCR in bladder cancer tissues and paracancerous tissues;
图2为实时荧光定量PCR检测piR-60152在膀胱癌组织及癌旁组织中的表达。Figure 2 shows the expression of piR-60152 in bladder cancer tissues and adjacent tissues detected by real-time fluorescent quantitative PCR.
具体实施方式detailed description
以下结合实施例对本发明进行详细的说明：本实施例在以本发明技术方案的前提下进行实施，给出了详细的实施方式和过程，但本发明的包含范围不限于下述实施例。下面的实施例中未注明的条件和方法等均按照常规进行。The present invention is described in detail below in conjunction with the examples: the present example is implemented under the premise of the technical solution of the present invention, and detailed implementation and process are provided, but the scope of the present invention is not limited to the following examples. The conditions and methods not indicated in the following examples were carried out according to routine.
实施例1：样本的收集及组织中RNA的提取Example 1: Collection of Samples and Extraction of RNA in Tissues
收集到19对新发的膀胱癌及其对应癌旁正常组织。组织来源于2010年1月至2012年6月，且经过医院病理诊断确认，所有研究对象均签署知情同意书。19 pairs of new bladder cancer and their corresponding adjacent normal tissues were collected. The tissues were obtained from January 2010 to June 2012, and were confirmed by pathological diagnosis in the hospital, and all research subjects signed the informed consent.
使用Trizol法提取组织中的总RNA：称取100mg的膀胱癌组织或者癌旁正常组织放于玻璃研磨器中，继而加入液氮研磨至组织变成干粉状，而后将干粉用刮子转移至1.5mL去RNA酶的离心管中（管中提前加好1mLTrizol溶液），颠倒混匀10下，室温静置5分钟；每1mLTrizol中加入0.2mL氯仿，盖紧样品管盖，用手用力摇晃试管15秒，使其充分混匀，室温静置5分钟后12000g离心15分钟；将上层水相转入新的1.5mL去RNA酶的离心管中（上清量约0.4-0.5mL），加入0.5mL异丙醇，混匀后放于-20℃中1小时，然后12000g离心10分钟；小心倒掉上清液，留取离心管底部的沉淀，加1mL现配的75%wt的乙醇（预冷）振荡洗涤RNA沉淀一次，最后7500g离心5分钟；小心倒掉上清，而后将带有沉淀的离心管置于超净工作台开风机吹干（约30分钟，此时RNA沉淀变透明）；在离心管中加20μL的DEPC水溶解，可在55-60℃下孵育10分钟助溶。提取好的总RNA保存于-70℃的超低温冰箱中，或立即用于逆转录。总RNA质量分析：通过对RNAA230、A260及A280的检测（A260/A280=1.8-2.1,A260/A230>1.80;），以及对总RNA采用琼脂糖凝胶电泳，根据28SrRNA与18SrRNA的比值分析总RNA的质量。Use the Trizol method to extract total RNA from tissues: Weigh 100 mg of bladder cancer tissue or adjacent normal tissue and put it in a glass grinder, then add liquid nitrogen to grind until the tissue becomes a dry powder, and then transfer the dry powder to the In a 1.5mL RNase-removing centrifuge tube (1mL Trizol solution was added to the tube in advance), invert and mix for 10 times, and let stand at room temperature for 5 minutes; add 0.2mL chloroform to each 1mL Trizol, cover the sample tube tightly, and shake the test tube vigorously by hand Mix well for 15 seconds, let stand at room temperature for 5 minutes and then centrifuge at 12000g for 15 minutes; Mix 1 mL of isopropanol, put it at -20°C for 1 hour, then centrifuge at 12,000 g for 10 minutes; pour off the supernatant carefully, keep the sediment at the bottom of the centrifuge tube, add 1 mL of 75%wt ethanol (pre- Cold) Shake and wash the RNA pellet once, and finally centrifuge at 7500g for 5 minutes; pour off the supernatant carefully, and then place the centrifuge tube with the pellet on the ultra-clean bench to blow dry (about 30 minutes, when the RNA pellet becomes transparent) ;Add 20μL of DEPC water to the centrifuge tube to dissolve, and incubate at 55-60℃ for 10 minutes to help dissolve. The extracted total RNA was stored in a -70°C ultra-low temperature freezer, or used for reverse transcription immediately. Total RNA quality analysis: through the detection of RNA A230, A260 and A280 (A260/A280=1.8-2.1, A260/A230>1.80;), and agarose gel electrophoresis for total RNA, the total RNA was analyzed according to the ratio of 28SrRNA to 18SrRNA RNA quality.
样本流行病学及临床资料的调查Survey of sample epidemiology and clinical data
所有患者的膀胱组织都经过两名病理医生的病理诊断，纳入本研究前未患其它肿瘤，且未经放射或化学药物治疗。调查内容包括一般人口学特征、临床资料、吸烟史、饮酒史、饮食、个人史和家族史等。吸烟者定义为每天吸烟超过1支并持续一年以上，累计吸烟量“包年”按(每日吸烟量/20)×吸烟年数来计算。饮酒者定义为每周至少3次，持续一年以上。The bladder tissues of all patients had been pathologically diagnosed by two pathologists, and they had no other tumors before inclusion in this study, and had not received radiation or chemical drug treatment. The survey content included general demographic characteristics, clinical data, smoking history, drinking history, diet, personal history and family history. A smoker is defined as smoking more than one cigarette per day for more than one year, and the cumulative smoking volume "package year" is calculated by (daily smoking volume/20) x smoking years. Drinkers were defined as at least 3 times per week for more than one year.
piRNA表达谱用于筛选膀胱癌及其对应癌旁正常组织中差异表达的候选piRNApiRNA expression profiling for screening candidate piRNAs differentially expressed in bladder cancer and its corresponding paracancerous normal tissues
随机抽取3对癌及癌旁正常组织，采用人piRNA芯片检测了23677个piRNAs。统计学分析结果显示：差异表达2倍以上且P值小于0.05的piRNA基因数为197个，其中上调的106个，下调的91个（表1）。我们列出了差异表达上调、下调最显著的前十个piRNAs（表2）。Three pairs of cancer and adjacent normal tissues were randomly selected, and 23677 piRNAs were detected by human piRNA chip. The results of statistical analysis showed that there were 197 piRNA genes with differential expression of more than 2 times and P value less than 0.05, including 106 up-regulated and 91 down-regulated (Table 1). We listed the top ten piRNAs with the most significant up- and down-regulation in differential expression (Table 2).
膀胱癌组织及其癌旁正常组织中piRNA表达量的检测方法Detection method of piRNA expression in bladder cancer tissue and its adjacent normal tissue
采用SYBRqRT-PCR的方法检测膀胱组织中piRNA的表达量Using SYBRqRT-PCR to detect the expression of piRNA in bladder tissue
（1）逆转录：采用miScript逆转录试剂盒（Qiagen）对提取出的总RNA进行逆转录反应，具体操作依据该试剂盒的说明书进行。首先在0.5mL离心管中加入5μLDEPC双蒸水、继而加入1μL逆转录混合试剂和4μLRT缓冲液，最后加入10μL0.1μg/μL的总RNA，总反应体系20μL。该方法的反应条件为：37℃反应1小时，95℃反应5分钟，得到cDNA溶液。(1) Reverse transcription: The extracted total RNA was reverse-transcribed using miScript Reverse Transcription Kit (Qiagen), and the specific operation was carried out according to the instructions of the kit. First add 5 μL DEPC double-distilled water to a 0.5 mL centrifuge tube, then add 1 μL reverse transcription mixed reagent and 4 μL RT buffer, and finally add 10 μL 0.1 μg/μL total RNA, the total reaction system is 20 μL. The reaction conditions of the method are: react at 37° C. for 1 hour, and react at 95° C. for 5 minutes to obtain a cDNA solution.
（2）piR-52681实时荧光定量PCR检测：采用miScriptSYBRGreenPCR检测试剂盒对（1）中获得的cDNA溶液进行PCR扩增反应，扩增引物为piR-52681扩增上游引物和PCR检测的小RNA通用的下游引物，再用ABI7900HTReal-TimePCR仪检测，具体操作依据该试剂盒与荧光定量PCR仪说明书进行。反应体系包括：1μLpiR-52681扩增上游引物和1μLPCR检测的小RNA通用的下游引物，5μLSYBRGreenPCR反应缓冲液，2μLDEPC双蒸水以及1μLcDNA稀释液（0.1μg/μL）。该方法的反应条件：95℃预变性15分钟，94℃变性15秒，60℃退火30秒，70℃延伸30秒，94℃-70℃的阶段共40个循环。(2) Real-time fluorescent quantitative PCR detection of piR-52681: Use miScriptSYBRGreenPCR detection kit to perform PCR amplification reaction on the cDNA solution obtained in (1). The downstream primers were detected with the ABI7900HTReal-Time PCR instrument, and the specific operation was carried out according to the instructions of the kit and the fluorescent quantitative PCR instrument. The reaction system includes: 1 μL piR-52681 amplification upstream primer and 1 μL PCR detection small RNA universal downstream primer, 5 μL SYBRGreen PCR reaction buffer, 2 μL DEPC double distilled water and 1 μL cDNA diluent (0.1 μg/μL). The reaction conditions of this method are: 95°C pre-denaturation for 15 minutes, 94°C denaturation for 15 seconds, 60°C annealing for 30 seconds, 70°C extension for 30 seconds, 40 cycles of 94°C-70°C stages.
（3）piR-60152实时荧光定量PCR检测：与（2）中的方法基本相同，所不同的只是扩增的上游引物由piR-52681特异性扩增上游引物代替piR-60152，检测得到膀胱癌组织及癌旁组织中piR-60152的表达水平。(3) piR-60152 real-time fluorescence quantitative PCR detection: the method is basically the same as in (2), the difference is that the amplified upstream primer is specifically amplified by piR-52681 instead of piR-60152, and bladder cancer is detected Expression levels of piR-60152 in tissues and adjacent tissues.
（4）U6实时荧光定量PCR检测：与（2）中的方法基本相同，所不同的只是扩增的上游引物有piR-52681特异性扩增上游引物代替U6，检测得到膀胱癌组织及癌旁组织中U6的表达水平。在本实验中U6作为piR-52681与piR-60152的内参。(4) U6 real-time fluorescence quantitative PCR detection: the method is basically the same as in (2), the difference is that the amplified upstream primer has piR-52681 specific amplification upstream primer instead of U6, and the bladder cancer tissue and adjacent tumors can be detected Expression levels of U6 in tissues. In this experiment, U6 was used as an internal reference for piR-52681 and piR-60152.
分析piR-52681（DQ585569）和piR-60152（DQ594040）对膀胱癌的诊断价值Analysis of the diagnostic value of piR-52681 (DQ585569) and piR-60152 (DQ594040) in bladder cancer
实例中piRNA的表达是以CT值来表示。CT值代表一种目的piRNA分子达到实验设计阈值所需要的PCR循环的次数。在目的piR-52681、piR-60152和内参U6扩增效率相同的情况下，可以直接得到目的piR-52681、piR-60152相当于内参的定量ΔCT=CT目的-CT内参。采用2-ΔCT表示目的piRNA表达相对于内参U6表达变化的倍数。所有的统计检验均为双侧概率检验，P<0.05为差异有统计学意义。采用SPSS13.0软件做进一步的统计分析。The expression of piRNA in the example is represented by CT value. The CT value represents the number of PCR cycles required for a target piRNA molecule to reach the threshold of the experimental design. In the case that the target piR-52681, piR-60152 and the internal reference U6 have the same amplification efficiency, the quantitative ΔCT=CTtarget -CTinternal reference of the target piR-52681 and piR-60152 equivalent to the internal reference can be directly obtained. 2-ΔCT is used to represent the fold change of the target piRNA expression relative to the expression of the internal reference U6. All statistical tests were two-sided probability tests, and P<0.05 was considered statistically significant. SPSS13.0 software was used for further statistical analysis.
统计分析结果发现，piR-52681在膀胱癌组织中的表达显著高于其对应的癌旁正常组织（癌组织与癌旁正常组织相比：7.58±7.49versus2.91±1.33,P=0.026）（图1）。piR-60152在膀胱癌组织中的表达显著低于其对应的癌旁正常组织（癌组织与癌旁正常组织相比：3.95±2.05versus8.82±7.81,P=0.027）（图2）。Statistical analysis showed that the expression of piR-52681 in bladder cancer tissues was significantly higher than that in the corresponding normal adjacent tissues (compared between cancerous tissues and adjacent normal tissues: 7.58±7.49versus2.91±1.33, P=0.026) ( figure 1). The expression of piR-60152 in bladder cancer tissues was significantly lower than that in its corresponding adjacent normal tissues (compared with cancer tissues and adjacent normal tissues: 3.95±2.05versus8.82±7.81, P=0.027) (Figure 2).
表1.膀胱癌及癌旁正常组织中差异piRNA表达谱Table 1. Differential piRNA expression profiles in bladder cancer and adjacent normal tissues
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aNCBI数据库a NCBI database
表2.差异表达上调、下调最显著的前十个piRNAsTable 2. The top ten piRNAs with the most significant up-regulation and down-regulation in differential expression
aNCBI数据库a NCBI database
上述实例只为说明本发明的技术构思及特点，其目的在于让熟悉此项技术的人是能够了解本发明的内容并据以实施，并不能以此限制本发明的保护范围。凡根据本发明精神实质所做的等效变换或修饰，都应涵盖在本发明的保护范围之内。The above examples are only to illustrate the technical conception and characteristics of the present invention, and its purpose is to allow people familiar with this technology to understand the content of the present invention and implement it accordingly, and cannot limit the protection scope of the present invention. All equivalent changes or modifications made according to the spirit of the present invention shall fall within the protection scope of the present invention.
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<213>人工序列<213> Artificial sequence
<400>41<400>41
ctgtggagaagagatcctgatcacggtgct30ctgtggagaagagatcctgatcacggtgct30
<210>42<210>42
<211>26<211>26
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>42<400>42
caaagatgagtggtgaaaatctgatc26caaagatgagtggtgaaaatctgatc26
<210>43<210>43
<211>30<211>30
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>43<400>43
ctcacaaagatgagtggtgaaaatctgatc30ctcacaaagatgagtggtgaaaatctgatc30
<210>44<210>44
<211>26<211>26
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>44<400>44
tggtgagctgcgacaactggatgaac26tggtgagctgcgacaactggatgaac26
<210>45<210>45
<211>26<211>26
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>45<400>45
cggctgttaaccgaaaggttggtggt26cggctgttaaccgaaaggttggtggt26
<210>46<210>46
<211>26<211>26
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>46<400>46
gtcagggacatttctgaagcgagcgt26gtcagggaacatttctgaagcgagcgt26
<210>47<210>47
<211>26<211>26
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>47<400>47
tgaagcagaggcaatgaattacgaag26tgaagcagaggcaatgaattacgaag26
<210>48<210>48
<211>28<211>28
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>48<400>48
tcgctggttcgaatccggctcggaggac28tcgctggttcgaatccggctcggaggac28
<210>49<210>49
<211>29<211>29
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>49<400>49
tgtcacgtgtgtggcaagatgctgagctc29tgtcacgtgtgtggcaagatgctgagctc29
<210>50<210>50
<211>31<211>31
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>50<400>50
gttaagatggcagagcccggtaatcgcataa31gttaagatggcagagcccggtaatcgcataa31
<210>51<210>51
<211>30<211>30
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>51<400>51
ttcgatgaagagatgatgacgagtctgact30ttcgatgaagagatgatgacgagtctgact30
<210>52<210>52
<211>30<211>30
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>52<400>52
gaggagccactgtggcgacagaatgctagc30gaggagccactgtggcgacagaatgctagc30
<210>53<210>53
<211>27<211>27
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>53<400>53
tgaagctgcagaaccaacgaggtggcc27tgaagctgcagaaccaacgaggtggcc27
<210>54<210>54
<211>27<211>27
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>54<400>54
tgggaagtacgtcctggggtacaagca27tgggaagtacgtcctggggtacaagca27
<210>55<210>55
<211>32<211>32
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>55<400>55
tcctaggactgaatcaccaccccagaagagca32tcctagactgaatcaccaccccagaagagca32
<210>56<210>56
<211>30<211>30
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>56<400>56
agcacatgatgattccagcgtgcgtctgaa30agcacatgatgattccagcgtgcgtctgaa30
<210>57<210>57
<211>27<211>27
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>57<400>57
tgcaatggcatgatctcggctcactgc27tgcaatggcatgatctcggctcactgc27
<210>58<210>58
<211>32<211>32
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>58<400>58
cgtgctgggcccataacccagaggtcgatgga32cgtgctgggcccataacccagaggtcgatgga32
<210>59<210>59
<211>30<211>30
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>59<400>59
tcccacgtggctgagactacaggtgcttgc30tcccacgtggctgagactacaggtgcttgc30
<210>60<210>60
<211>30<211>30
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>60<400>60
tacctgaagatgaaaggggactactaccgc30tacctgaagatgaaaggggactactaccgc30
<210>61<210>61
<211>28<211>28
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>61<400>61
tgcagaaagctctgggtgtgcggcagta28tgcagaaagctctgggtgtgcggcagta28
<210>62<210>62
<211>28<211>28
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>62<400>62
ttggaggatgaaacaaaggaatctgact28ttggaggatgaaacaaaggaatctgact28
<210>63<210>63
<211>30<211>30
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>63<400>63
tgccaccaatgtcgagcaggcgttcatgac30tgccaccaatgtcgagcaggcgttcatgac30
<210>64<210>64
<211>27<211>27
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>64<400>64
tgactgtgctgtcctgattgttgctgc27tgactgtgctgtcctgattgttgctgc27
<210>65<210>65
<211>29<211>29
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>65<400>65
acaaaaaattacccgggcgtggtggcagg29acaaaaaattacccgggcgtggtggcagg29
<210>66<210>66
<211>26<211>26
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>66<400>66
tggagatcctggagctgtactgtgac26tggagatcctggagctgtactgtgac26
<210>67<210>67
<211>30<211>30
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>67<400>67
tctcaaagtgctgggattacaggcgtgagc30tctcaaagtgctgggattacaggcgtgagc30
<210>68<210>68
<211>30<211>30
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>68<400>68
tcctaaagtgctggggttacaggcatgagc30tcctaaagtgctggggttacaggcatgagc30
<210>69<210>69
<211>31<211>31
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>69<400>69
tgctagggtcacctgtaccccaggctggagc31tgctagggtcacctgtaccccaggctggagc31
<210>70<210>70
<211>28<211>28
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>70<400>70
tgatgagacgaagttgacgctgcatgga28tgatgagacgaagttgacgctgcatgga28
<210>71<210>71
<211>26<211>26
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>71<400>71
tggtggatgcgcacactgccgaagtc26tggtggatgcgcacactgccgaagtc26
<210>72<210>72
<211>30<211>30
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>72<400>72
tagcagctgtatcgggcttcctcatcctgc30tagcagctgtatcgggcttcctcatcctgc30
<210>73<210>73
<211>30<211>30
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>73<400>73
tggtctcaaactcctgacctcaggtgatct30tggtctcaaactcctgacctcaggtgatct30
<210>74<210>74
<211>31<211>31
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>74<400>74
tagggtcacctgtaccccaggctggagctgc31tagggtcacctgtaccccaggctggagctgc31
<210>75<210>75
<211>27<211>27
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>75<400>75
tcggaacctgcagacacttgtggagga27tcggaacctgcagacacttgtggagga27
<210>76<210>76
<211>27<211>27
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>76<400>76
ccccccactgctaaatttgactggcta27ccccccactgctaaatttgactggcta27
<210>77<210>77
<211>30<211>30
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>77<400>77
tgctgggatgacaggcatgagccactgcgc30tgctgggatgacaggcatgagccactgcgc30
<210>78<210>78
<211>28<211>28
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>78<400>78
tcccaaagtgctggaattacaggcgtga28tcccaaagtgctggaattacaggcgtga28
<210>79<210>79
<211>29<211>29
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>79<400>79
tggtctcgaactcctgacctcaggtgatc29tggtctcgaactcctgacctcaggtgatc29
<210>80<210>80
<211>28<211>28
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>80<400>80
tagtttcagaaggaatggtaccagctcc28tagtttcagaaggaatggtaccagctcc28
<210>81<210>81
<211>26<211>26
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>81<400>81
tacagcacactgatgggtcttgactc26tacagcacactgatgggtcttgactc26
<210>82<210>82
<211>29<211>29
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>82<400>82
tcacaaagatgagtggtgaaaatctgatc29tcacaaagatgagtggtgaaaatctgatc29
<210>83<210>83
<211>32<211>32
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>83<400>83
tccatcgttacaatggcctctttagacccagc32tccatcgttacaatggcctctttagacccagc32
<210>84<210>84
<211>31<211>31
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>84<400>84
tccgagtgcagaccacgccggactacagccc31tccgagtgcagaccacgccggactacagccc31
<210>85<210>85
<211>30<211>30
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>85<400>85
tacctcatgaagatcctcaccgagcgcggc30tacctcatgaagatcctcaccgagcgcggc30
<210>86<210>86
<211>30<211>30
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>86<400>86
tggtctcgaactcctgacctcaggtgatcc30tggtctcgaactcctgacctcaggtgatcc30
<210>87<210>87
<211>30<211>30
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>87<400>87
tggtgtgatctcggctcactgcaacctcct30tggtgtgatctcggctcactgcaacctcct30
<210>88<210>88
<211>30<211>30
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>88<400>88
tcgcaatccaactggtacactggcatgagt30tcgcaatccaactggtacactggcatgagt30
<210>89<210>89
<211>30<211>30
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>89<400>89
cgatcaagcagccgtgcagagatgtgcctc30cgatcaagcagccgtgcagagatgtgcctc30
<210>90<210>90
<211>29<211>29
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>90<400>90
tgggattacaggcatgagccaccgcgcct29tgggattacaggcatgagccaccgcgcct29
<210>91<210>91
<211>30<211>30
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>91<400>91
tggtctcgaactcctgacctcaggtgatct30tggtctcgaactcctgacctcaggtgatct30
<210>92<210>92
<211>30<211>30
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>92<400>92
tcccaaagtgctgggaatacaggcatgagc30tcccaaagtgctgggaatacaggcatgagc30
<210>93<210>93
<211>30<211>30
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>93<400>93
ctcgaactcctgacctcaggtgatctgcct30ctcgaactcctgacctcaggtgatctgcct30
<210>94<210>94
<211>31<211>31
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>94<400>94
tggtctcgaactcctgacctcaggtgatcca31tggtctcgaactcctgacctcaggtgatcca31
<210>95<210>95
<211>26<211>26
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>95<400>95
tggtctcgaactcctgacctcaggta26tggtctcgaactcctgacctcaggta26
<210>96<210>96
<211>29<211>29
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>96<400>96
gagaaagctcacaagaactgctaactcat29gagaaagctcacaagaactgctaactcat29
<210>97<210>97
<211>28<211>28
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>97<400>97
tgggctgcggtggccagggccgtgagtc28tgggctgcggtggccagggccgtgagtc28
<210>98<210>98
<211>26<211>26
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>98<400>98
catttgcggccggcgccagggtggag26catttgcggccggcgccagggtggag26
<210>99<210>99
<211>28<211>28
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>99<400>99
tgggaacctgcagacacttgtggaggag28tgggaacctgcagacacttgtggaggag28
<210>100<210>100
<211>30<211>30
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>100<400>100
gagaaagctcacaagaactgctaactcaca30gagaaagctcacaagaactgctaactcaca30
<210>101<210>101
<211>28<211>28
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>101<400>101
ctcctgaagtcagcgagaccacgaaccc28ctcctgaagtcagcgagaccacgaaccc28
<210>102<210>102
<211>26<211>26
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>102<400>102
tgtgtgctaaatgtgttcgtgacagg26tgtgtgctaaatgtgttcgtgacagg26
<210>103<210>103
<211>26<211>26
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>103<400>103
ttggaacaaccactgagctacggaga26ttggaacaaccactgagctacggaga26
<210>104<210>104
<211>30<211>30
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>104<400>104
tgatcaagcagccgtgcagagatgtgcctc30tgatcaagcagccgtgcagagatgtgcctc30
<210>105<210>105
<211>30<211>30
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>105<400>105
tgcctttgctgatctgacaggaggcagagc30tgcctttgctgatctgacaggaggcagagc30
<210>106<210>106
<211>30<211>30
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>106<400>106
taccatcttggctcactgcaacctccgcct30taccatcttggctcactgcaacctccgcct30
<210>107<210>107
<211>31<211>31
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>107<400>107
ttcaagccaggaaagcactgtagctccccca31ttcaagccaggaaagcactgtagctccccca31
<210>108<210>108
<211>27<211>27
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>108<400>108
gaccaatgatgagacagtgtttatgaa27gaccaatgatgagacagtgtttatgaa27
<210>109<210>109
<211>26<211>26
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>109<400>109
accaatgatgagacagtgtttatgaa26accaatgatgagacagtgtttatgaa26
<210>110<210>110
<211>28<211>28
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>110<400>110
gaccaatgatgagacagtgtttatgaaa28gaccaatgatgagacagtgtttatgaaa28
<210>111<210>111
<211>30<211>30
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>111<400>111
tggccccttacccttccagagatgaagccc30tggccccttacccttccagagatgaagccc30
<210>112<210>112
<211>28<211>28
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>112<400>112
gaccaatgatgagacagtgtttatgaac28gaccaatgatgagacagtgtttatgaac28
<210>113<210>113
<211>31<211>31
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>113<400>113
tccccaaaacaagcacagtagcctgcacagt31tccccaaaacaagcacagtagcctgcacagt31
<210>114<210>114
<211>26<211>26
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>114<400>114
tgcctgatgatctgagatggaacggt26tgcctgatgatctgagatggaacggt26
<210>115<210>115
<211>31<211>31
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>115<400>115
gaccaatgatgagtattctggggtgtctgaa31gaccaatgatgagtattctggggtgtctgaa31
<210>116<210>116
<211>30<211>30
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>116<400>116
gaccaatgatgagtattctggggtgtctga30gaccaatgatgagtattctggggtgtctga30
<210>117<210>117
<211>26<211>26
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>117<400>117
tcctcacaccgcacggcaggcacaga26tcctcacaccgcacggcaggcacaga26
<210>118<210>118
<211>29<211>29
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>118<400>118
tataccgagacattccattgcccagggac29tataccgagacattccattgcccagggac29
<210>119<210>119
<211>31<211>31
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>119<400>119
gacctatgatgatgactggtggcgtatgagt31gacctatgatgatgactggtggcgtatgagt31
<210>120<210>120
<211>31<211>31
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>120<400>120
tgagatgctgatggggatctatgaacggatc31tgagatgctgatggggatctatgaacggatc31
<210>121<210>121
<211>30<211>30
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>121<400>121
gctgttatgcatctaccggcgcccaccctc30gctgttatgcatctaccggcgcccaccctc30
<210>122<210>122
<211>27<211>27
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>122<400>122
tgggcaaggcagccagacaggcagtga27tgggcaaggcagccagacaggcagtga27
<210>123<210>123
<211>28<211>28
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>123<400>123
taactgaacttaaaagaaaagccaggga28taactgaacttaaaagaaaagccaggga28
<210>124<210>124
<211>27<211>27
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>124<400>124
tgactaattagactgggaaacaagggc27tgactaattagactgggaaacaagggc27
<210>125<210>125
<211>29<211>29
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>125<400>125
tgaggtgtaagcactgtatcaagacaggt29tgaggtgtaagcactgtatcaagacaggt29
<210>126<210>126
<211>31<211>31
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>126<400>126
gaccaatgatgagattggagggtgtctgaat31gaccaatgatgagattggagggtgtctgaat31
<210>127<210>127
<211>27<211>27
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>127<400>127
tgggcaagaagcactgtctgcaaggcc27tgggcaagaagcactgtctgcaaggcc27
<210>128<210>128
<211>30<211>30
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>128<400>128
gaccaatgatgagattggagggtgtctgaa30gaccaatgatgagattggagggtgtctgaa30
<210>129<210>129
<211>31<211>31
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>129<400>129
taccaatgatgagattggagggtgtctgaat31taccaatgatgagattggagggtgtctgaat31
<210>130<210>130
<211>30<211>30
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>130<400>130
tgtggcctgcaaaggaaggtaagtaggtgg30tgtggcctgcaaaggaaggtaagtaggtgg30
<210>131<210>131
<211>30<211>30
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>131<400>131
accaatgatgagattggagggtgtctgaat30accaatgatgagattggagggtgtctgaat30
<210>132<210>132
<211>26<211>26
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>132<400>132
tacagctcccgtgtctgggaagaagc26tacagctcccgtgtctgggaagaagc26
<210>133<210>133
<211>29<211>29
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>133<400>133
ctgcactgtagcctgggtgaccagagtga29ctgcactgtagcctgggtgaccagagtga29
<210>134<210>134
<211>29<211>29
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>134<400>134
taggtacaacggattccggcgggcagagc29taggtacaacggattccggcgggcagagc29
<210>135<210>135
<211>29<211>29
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>135<400>135
tggggtcctgcacagatgggacgtggctt29tggggtcctgcacagatgggacgtggctt29
<210>136<210>136
<211>31<211>31
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>136<400>136
tcccttcccgacttgatgaaggcagagtagc31tcccttcccgacttgatgaaggcagagtagc31
<210>137<210>137
<211>32<211>32
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>137<400>137
tctgagaagccacaccaacctcgttccgggga32tctgagaagccacaccaacctcgttccgggga32
<210>138<210>138
<211>31<211>31
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>138<400>138
tgtgagatggagcaagatgaaggccacatgt31tgtgagatggagcaagatgaaggccacatgt31
<210>139<210>139
<211>27<211>27
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>139<400>139
tgatgatcctgagatgagaccaaactg27tgatgatcctgagatgagaccaaactg27
<210>140<210>140
<211>26<211>26
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>140<400>140
tgaggccccgggttcgatccccggca26tgaggccccgggttcgatccccggca26
<210>141<210>141
<211>30<211>30
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>141<400>141
tgccagggaaacagtctcaattttacactt30tgccagggaaacagtctcaattttacactt30
<210>142<210>142
<211>29<211>29
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>142<400>142
gcggtgtgacgagactcgcgtgggaagaa29gcggtgtgacgagactcgcgtgggaagaa29
<210>143<210>143
<211>28<211>28
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>143<400>143
ggcacagagcaggctaggtctgccgcga28ggcacagagcaggctaggtctgccgcga28
<210>144<210>144
<211>32<211>32
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>144<400>144
ctgagaagccacaccaacctcgttccggggat32ctgagaagccaccaacctcgttccggggat32
<210>145<210>145
<211>29<211>29
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>145<400>145
tgaagttggggccgagcaagaaagtgcga29tgaagttggggccgagcaagaaagtgcga29
<210>146<210>146
<211>30<211>30
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>146<400>146
caggctaatgggaaacggagcaaccacatg30caggctaatgggaaacggagcaaccacatg30
<210>147<210>147
<211>26<211>26
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>147<400>147
acagcagccagggatagcgaaggtgg26acagcagccagggatagcgaaggtgg26
<210>148<210>148
<211>26<211>26
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>148<400>148
tgcgcaggaagactgagatgaggacc26tgcgcaggaagactgagatgaggacc26
<210>149<210>149
<211>29<211>29
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>149<400>149
gtaggatgaaaacactggggctgaaatgc29gtaggatgaaaacactggggctgaaatgc29
<210>150<210>150
<211>30<211>30
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>150<400>150
gaagatggagcagttgctagagacgatggt30gaagatggagcagttgctagagacgatggt30
<210>151<210>151
<211>29<211>29
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>151<400>151
tgcgcaggaagactgagatgaggacccta29tgcgcaggaagactgagatgaggacccta29
<210>152<210>152
<211>29<211>29
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>152<400>152
gccatctgaggtgtcgacagagaaagaga29gccatctgaggtgtcgacagagaaagaga29
<210>153<210>153
<211>28<211>28
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>153<400>153
agctgaaaaaggagaaagggtcactgag28agctgaaaaaggagaaagggtcactgag28
<210>154<210>154
<211>30<211>30
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>154<400>154
tggtaggaatggatgatgatgctgcagagg30tggtaggaatggatgatgatgctgcagagg30
<210>155<210>155
<211>31<211>31
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>155<400>155
tccagggagaaaaataaaaggaagccataga31tccagggagaaaaataaaaggaagccataga31
<210>156<210>156
<211>28<211>28
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>156<400>156
tatagagaaagaaataaggggacccggg28tatagagaaagaaataaggggacccggg28
<210>157<210>157
<211>31<211>31
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>157<400>157
aggggaaacacaggaagtgtttaaggcatgc31aggggaaacacaggaagtgtttaaggcatgc31
<210>158<210>158
<211>31<211>31
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>158<400>158
caggagagtaggatgaaaacactggggcttt31caggagagtaggatgaaaacactggggcttt31
<210>159<210>159
<211>30<211>30
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>159<400>159
gacctcaagggaatgtagagaagcctcagg30gacctcaagggaatgtagagaagcctcagg30
<210>160<210>160
<211>27<211>27
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>160<400>160
agcgcaggaagactgagatgaggaccc27agcgcaggaagactgagatgaggaccc27
<210>161<210>161
<211>31<211>31
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>161<400>161
gtggaaagctgagtaactgctgagtaggtgt31gtggaaagctgagtaactgctgagtaggtgt31
<210>162<210>162
<211>30<211>30
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>162<400>162
tctcatgggagtgcttaccaatgtgtggcc30tctcatgggagtgcttaccaatgtgtggcc30
<210>163<210>163
<211>30<211>30
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>163<400>163
cagcagagcaggcacagcggtgtgacgaga30cagcagagcaggcacagcggtgtgacgaga30
<210>164<210>164
<211>31<211>31
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>164<400>164
tcttgagggacgctagaggtagtagatgtgt31tcttgagggacgctagaggtagtagtagatgtgt31
<210>165<210>165
<211>30<211>30
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>165<400>165
gacatgcatagagggaagaccaggtgaaga30gacatgcatagagggaagaccaggtgaaga30
<210>166<210>166
<211>27<211>27
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>166<400>166
aggagaagtagaaggggctgcacatgc27aggagaagtagaaggggctgcacatgc27
<210>167<210>167
<211>29<211>29
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>167<400>167
tcggcagagcaggcacagaggtgtgacga29tcggcagagcaggcacagaggtgtgacga29
<210>168<210>168
<211>26<211>26
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>168<400>168
tggagacacaggacgttctgagatgg26tggagacacaggacgttctgagatgg26
<210>169<210>169
<211>26<211>26
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>169<400>169
tgtgaaggaaagtcaccacagggcaa26tgtgaaggaaagtcaccacagggcaa26
<210>170<210>170
<211>29<211>29
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>170<400>170
tgatggagagccctgtgctgagatgaaat29tgatggagagccctgtgctgagatgaaat29
<210>171<210>171
<211>26<211>26
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>171<400>171
tgatagtggaactaaaatcgggggca26tgatagtggaactaaaatcgggggca26
<210>172<210>172
<211>30<211>30
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>172<400>172
tagagagtcagaagctctatatgcgtagtg30tagagagtcagaagctctatatgcgtagtg30
<210>173<210>173
<211>30<211>30
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>173<400>173
actgaatgaaaagaggtggctgaggaaagc30actgaatgaaaagaggtggctgaggaaagc30
<210>174<210>174
<211>31<211>31
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>174<400>174
tattgagtggaagtagctagggtctcagggg31tattgagtggaagtagctagggtctcagggg31
<210>175<210>175
<211>28<211>28
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>175<400>175
tgcaggacaaagacaatctggctaggga28tgcaggacaaagacaatctggctaggga28
<210>176<210>176
<211>29<211>29
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>176<400>176
tctcctgatgccttgttatgaacagggag29tctcctgatgccttgttatgaacagggag29
<210>177<210>177
<211>28<211>28
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>177<400>177
tccaagaaatactgggagtgctggaccc28tccaagaaatactgggagtgctggaccc28
<210>178<210>178
<211>26<211>26
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>178<400>178
gcagagcaagaaaagcagagatgtgg26gcagagcaagaaaagcagagatgtgg26
<210>179<210>179
<211>31<211>31
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>179<400>179
gcagagcaggcacagcggtgcgacgagatgt31gcagagcaggcacagcggtgcgacgagatgt31
<210>180<210>180
<211>29<211>29
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>180<400>180
taggagtacggtctaggcaaaggcatggt29taggagtacggtctaggcaaaggcatggt29
<210>181<210>181
<211>26<211>26
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>181<400>181
taggaccagagatggagaatgacagc26taggaccagagatggagaatgacagc26
<210>182<210>182
<211>30<211>30
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>182<400>182
tagaaggtgagatctggagttaaacatgtc30tagaaggtgagatctggagttaaacatgtc30
<210>183<210>183
<211>26<211>26
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>183<400>183
ggaaaaaaagcaagatgcctcaatgc26ggaaaaaaagcaagatgcctcaatgc26
<210>184<210>184
<211>28<211>28
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>184<400>184
cgcgtgggaacgagaagacactcgtgga28cgcgtgggaacgagaagaacactcgtgga28
<210>185<210>185
<211>28<211>28
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>185<400>185
tacatcatggaccaaaggacagacaggc28tacatcatggaccaaaggacagacaggc28
<210>186<210>186
<211>28<211>28
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>186<400>186
tgggagaaaagccaagcaagtgctgaac28tgggagaaaagccaagcaagtgctgaac28
<210>187<210>187
<211>27<211>27
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>187<400>187
cgccaaaggatagaaccgtcaggggcc27cgccaaaggatagaaccgtcaggggcc27
<210>188<210>188
<211>28<211>28
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>188<400>188
taggatgaaaacactggggctgaaatgc28taggatgaaaacactggggctgaaatgc28
<210>189<210>189
<211>30<211>30
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>189<400>189
ggaactcggccaggtgctgatcggtgaggt30ggaactcggccaggtgctgatcggtgaggt30
<210>190<210>190
<211>26<211>26
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>190<400>190
tgctgagatggagaagaccgggggca26tgctgagatggagaagaccggggggca26
<210>191<210>191
<211>29<211>29
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>191<400>191
ggcaaactggtggagatgtgaactgaggc29ggcaaactggtggagatgtgaactgaggc29
<210>192<210>192
<211>29<211>29
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>192<400>192
gaggaaggcagaacaaaggaaataagaag29gaggaaggcagaacaaaggaaataagaag29
<210>193<210>193
<211>28<211>28
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>193<400>193
cggcggtggcggcggcggcggcgggacc28cggcggtggcggcggcggcggcgggacc28
<210>194<210>194
<211>30<211>30
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>194<400>194
gaagctatgggagataagatggacttcctt30gaagctatgggagataagatggacttcctt30
<210>195<210>195
<211>30<211>30
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>195<400>195
tggaggaactcaccttccagggtgtcaatg30tggaggaactcaccttccagggtgtcaatg30
<210>196<210>196
<211>31<211>31
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>196<400>196
tgtgaagttgcccacccccatgcagagcctt31tgtgaagttgcccacccccatgcagagcctt31
<210>197<210>197
<211>29<211>29
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<400>197<400>197
gaccaaggagttcatcttctcagagctgc29gaccaaggagttcatcttctcagagctgc29
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310516529.4ACN103627705B (en) | 2013-10-28 | 2013-10-28 | The piRNA biomarker relevant to bladder cancer and application thereof |
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310516529.4ACN103627705B (en) | 2013-10-28 | 2013-10-28 | The piRNA biomarker relevant to bladder cancer and application thereof |
| Publication Number | Publication Date |
|---|---|
| CN103627705A CN103627705A (en) | 2014-03-12 |
| CN103627705Btrue CN103627705B (en) | 2016-02-24 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310516529.4AExpired - Fee RelatedCN103627705B (en) | 2013-10-28 | 2013-10-28 | The piRNA biomarker relevant to bladder cancer and application thereof |
| Country | Link |
|---|---|
| CN (1) | CN103627705B (en) |
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| JP2019512014A (en)* | 2016-02-26 | 2019-05-09 | イェール ユニバーシティーＹａｌｅ Ｕｎｉｖｅｒｓｉｔｙ | Compositions and methods of using piRNA in cancer diagnostics and therapeutics |
| CN110546261B (en)* | 2017-03-29 | 2023-07-25 | 中国医学科学院基础医学研究所 | Small RNAs and their use in the prevention and/or treatment of fibroproliferative disorders and/or syndromes |
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| CN116694761B (en)* | 2023-04-29 | 2024-06-11 | 中国人民解放军总医院第二医学中心 | PiRNA marker for diagnosing breast cancer chemotherapy heart injury, kit and application thereof |
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| CN101289692B (en)* | 2008-05-22 | 2011-01-26 | 宁波大学 | A low-expression piRNA probe for detecting gastric tissue and its detection method |
| BR112012029656A2 (en)* | 2010-05-21 | 2016-12-13 | Uni Fur Bodenkultur Wien | compositions for use in the treatment or diagnosis of bone disorders and / or cardiovascular disorders |
| DK2631291T3 (en)* | 2010-10-22 | 2019-06-11 | Olix Pharmaceuticals Inc | NUCLEAR ACID MOLECULES INDUCING RNA INTERFERENCE AND USES THEREOF |
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