CN115011602A - 一种用于诊断急性呼吸窘迫综合征的circRNA标志物及检测试剂和试剂盒 - Google Patents
一种用于诊断急性呼吸窘迫综合征的circRNA标志物及检测试剂和试剂盒 Download PDFInfo
- Publication number
- CN115011602A CN115011602A CN202210770010.8A CN202210770010A CN115011602A CN 115011602 A CN115011602 A CN 115011602A CN 202210770010 A CN202210770010 A CN 202210770010A CN 115011602 A CN115011602 A CN 115011602A
- Authority
- CN
- China
- Prior art keywords
- circrna
- hsa
- marker
- artificial sequence
- ards
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 206010001052 Acute respiratory distress syndrome Diseases 0.000 title claims abstract description 54
- 201000000028 adult respiratory distress syndrome Diseases 0.000 title claims abstract description 52
- 208000013616 Respiratory Distress Syndrome Diseases 0.000 title claims abstract description 51
- 239000003550 marker Substances 0.000 title claims abstract description 24
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 13
- 238000001514 detection method Methods 0.000 title description 11
- 239000002773 nucleotide Substances 0.000 claims abstract description 12
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 12
- 210000001808 exosome Anatomy 0.000 abstract description 47
- 239000012530 fluid Substances 0.000 abstract description 22
- 238000002360 preparation method Methods 0.000 abstract description 3
- 108020004414 DNA Proteins 0.000 description 24
- 238000003753 real-time PCR Methods 0.000 description 9
- 238000000034 method Methods 0.000 description 8
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 7
- 241000282414 Homo sapiens Species 0.000 description 7
- 238000011529 RT qPCR Methods 0.000 description 7
- 108091028075 Circular RNA Proteins 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 6
- 238000010839 reverse transcription Methods 0.000 description 6
- 230000035945 sensitivity Effects 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 5
- 239000000523 sample Substances 0.000 description 5
- 206010069351 acute lung injury Diseases 0.000 description 4
- 230000003321 amplification Effects 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- 238000003745 diagnosis Methods 0.000 description 4
- 238000003199 nucleic acid amplification method Methods 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 238000004627 transmission electron microscopy Methods 0.000 description 4
- 238000011282 treatment Methods 0.000 description 4
- 238000012795 verification Methods 0.000 description 4
- 238000001262 western blot Methods 0.000 description 4
- 230000001154 acute effect Effects 0.000 description 3
- 239000000090 biomarker Substances 0.000 description 3
- 238000013276 bronchoscopy Methods 0.000 description 3
- 238000002955 isolation Methods 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000003762 quantitative reverse transcription PCR Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 206010002091 Anaesthesia Diseases 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 238000000692 Student's t-test Methods 0.000 description 2
- 108091046869 Telomeric non-coding RNA Proteins 0.000 description 2
- 230000037005 anaesthesia Effects 0.000 description 2
- 239000003146 anticoagulant agent Substances 0.000 description 2
- 229940127219 anticoagulant drug Drugs 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 210000004072 lung Anatomy 0.000 description 2
- 238000002844 melting Methods 0.000 description 2
- 230000008018 melting Effects 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 102100025222 CD63 antigen Human genes 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 208000002699 Digestive System Neoplasms Diseases 0.000 description 1
- 206010061818 Disease progression Diseases 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 108060002716 Exonuclease Proteins 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 101710088172 HTH-type transcriptional regulator RipA Proteins 0.000 description 1
- 101000934368 Homo sapiens CD63 antigen Proteins 0.000 description 1
- 101000613251 Homo sapiens Tumor susceptibility gene 101 protein Proteins 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 208000029523 Interstitial Lung disease Diseases 0.000 description 1
- NNJVILVZKWQKPM-UHFFFAOYSA-N Lidocaine Chemical compound CCN(CC)CC(=O)NC1=C(C)C=CC=C1C NNJVILVZKWQKPM-UHFFFAOYSA-N 0.000 description 1
- 239000002033 PVDF binder Substances 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 208000005718 Stomach Neoplasms Diseases 0.000 description 1
- 244000269722 Thea sinensis Species 0.000 description 1
- 102100040879 Tumor susceptibility gene 101 protein Human genes 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 210000001132 alveolar macrophage Anatomy 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 210000000621 bronchi Anatomy 0.000 description 1
- 230000008568 cell cell communication Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- -1 circRNA Chemical class 0.000 description 1
- 238000007621 cluster analysis Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000013075 data extraction Methods 0.000 description 1
- 230000005750 disease progression Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000003596 drug target Substances 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000000635 electron micrograph Methods 0.000 description 1
- 102000013165 exonuclease Human genes 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 206010017758 gastric cancer Diseases 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000013537 high throughput screening Methods 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 238000010191 image analysis Methods 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 229960004194 lidocaine Drugs 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 108091070501 miRNA Proteins 0.000 description 1
- 239000002679 microRNA Substances 0.000 description 1
- 238000002493 microarray Methods 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 239000003147 molecular marker Substances 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 239000000101 novel biomarker Substances 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 238000003921 particle size analysis Methods 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 239000013610 patient sample Substances 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000002685 pulmonary effect Effects 0.000 description 1
- 238000010814 radioimmunoprecipitation assay Methods 0.000 description 1
- 230000009711 regulatory function Effects 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 208000026425 severe pneumonia Diseases 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 201000011549 stomach cancer Diseases 0.000 description 1
- 230000003319 supportive effect Effects 0.000 description 1
- 238000005199 ultracentrifugation Methods 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- 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
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
-
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
-
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
本发明提供了一种circRNA,其核苷酸序列如SEQ ID NO.1所示。本发明提供了一种用于诊断急性呼吸窘迫综合征的circRNA标志物,所述circRNA标志物为hsa_circRNA_104034,所述的hsa_circRNA_104034核苷酸序列如SEQ ID NO.1所示。本发明还提供了circRNA标志物hsa_circRNA_104034在制备作为急性呼吸窘迫综合征检测标志物中的应用。本发明还提供了检测circRNA标志物hsa_circRNA_104034的试剂在制备用于诊断急性呼吸窘迫综合征的试剂盒中的应用。本发明发现ARDS的血浆和肺泡灌洗液外泌体hsa_circRNA_104034,可用来区分ARDS病人和正常人。
Description
技术领域
本发明属于生物检测领域,涉及急性呼吸窘迫综合征,具体来说是一种用于诊断急性呼吸窘迫综合征的circRNA标志物及检测试剂和试剂盒。
背景技术
急性肺损伤(Acute Lung Injury,ALI)及其更为严重阶段的急性呼吸窘迫综合征(Acute Respiratory Distress Syndrome,ARDS)是由多种病因引起的肺部急性弥漫性炎性损伤,是全球范围内严重威胁人类健康的高病死率急危重症;近期一项覆盖全球50个国家的大型流行病学调查数据显示,ARDS在ICU的发病率超过10%,死亡率高达34.9-46.1%。目前针对ALI/ARDS的治疗仍然集中在控制诱发ARDS的基础疾病及旨在改善气体交换和预防并发症为主的支持治疗。迄今尚无任何药物可以改善其预后,使ALI/ARDS的治疗面临巨大挑战。
外泌体介导的信息传递是细胞间通讯的新机制。外泌体是由多种细胞分泌的大小介于40-150nm的膜性囊泡,内含包括核酸(如circRNA、LncRNA、miRNA、DNA)、脂质、蛋白信号分子等多种成分,这些分子主要分为结构分子和亲代细胞特异性分子两大类。结构分子通常作为外泌体鉴定的分子标志物;亲代细胞特异性分子与外泌体来源的母细胞密切相关,在维持外泌体的独特生物功能方面发挥重要作用。不同细胞衍生的外泌体通过携带细胞活性成分到靶细胞调控其功能。
circRNA是具有反向剪接位点的闭合长链非编码RNA,近期研究表明circRNA在疾病进展中发挥重要的基因调节功能。与线性RNA分子相比,circRNA特有的闭合环状结构使其不易被RNA外切酶剪切,因而高度稳定,便于样本的保存;其次circRNA可以通过外泌体进入多种体液和分泌液,便于取样;circRNA长度多在200-400碱基之间,可用qPCR定量检测,可操作性强。因此,将circRNA用作疾病诊断的生物分子标志物具有明确的优势,近期在肝癌、胃癌等恶性消化系统肿瘤中相继发现了具有早期诊断价值的circRNA,但是在ARDS领域尚未见circRNA相关报道。
发明内容
针对现有技术中的上述技术问题,本发明提供了一种用于诊断急性呼吸窘迫综合征的circRNA标志物及检测试剂和试剂盒,所述的这种用于诊断急性呼吸窘迫综合征的circRNA标志物及检测试剂和试剂盒要解决现有技术中对于诊断急性呼吸窘迫综合征困难的技术问题。
本发明提供了一种circRNA,其核苷酸序列如SEQ ID NO.1所示。
本发明提供了一种用于诊断急性呼吸窘迫综合征的circRNA标志物,所述circRNA标志物为hsa_circRNA_104034,所述的hsa_circRNA_104034核苷酸序列如SEQ ID NO.1所示。
本发明还提供了circRNA标志物hsa_circRNA_104034在制备作为急性呼吸窘迫综合征检测标志物中的应用,所述的hsa_circRNA_104034核苷酸序列如SEQ ID NO.1所示。
本发明还提供了检测circRNA标志物hsa_circRNA_104034的试剂在制备用于诊断急性呼吸窘迫综合征的试剂盒中的应用,所述的hsa_circRNA_104034核苷酸序列如SEQ IDNO.1所示。
本发明还提供了一种试剂盒,其特征在于,包括检测circRNA标志物hsa_circRNA_104034的试剂,所述的hsa_circRNA_104034的核苷酸序列如SEQ ID NO.1所示。
进一步的,所述的试剂包括SEQ ID NO.2~23所示的引物。
本发明采用circRNA芯片检测以获取异常表达的肺泡灌洗液外泌体circRNA表达谱,并应用qPCR的方法进行验证。本发明发现ARDS的肺泡灌洗液外泌体hsa_circRNA_104034,可用来区分ARDS病人和正常人。扩大样本量验证hsa_circRNA_104034在患者肺泡灌洗液外泌体中的表达差异,发现在ARDS急性期,hsa_circRNA_104034在肺泡灌洗液外泌体的表达均明显升高,其诊断ARDS的敏感度是85%,特异度是90%,ROC曲线下的面积是1。
本发明还发现血浆hsa_circRNA_104034,可用来区分ARDS病人和正常人。发现在ARDS急性期,hsa_circRNA_104034在血浆中的表达明显升高。血浆hsa_circRNA_104034ROC曲线下的面积是0.799,敏感性71.05%,特异性78.95%。血浆hsa_circRNA_104034对ARDS的诊断具有较高的特异性和敏感度。
本发明和已有技术相比,其技术进步是显著的。本发明所提供的circRNA生物标志物对于人ARDS具有很高的灵敏度和特异性,可以作为新型的生物标志物用于ARDS的检测。本发明为临床医生快速判断ARDS患者的病情,为提高这类患者的临床治疗效果奠定基础,为发现具有潜在治疗价值的新型小分子药物靶标提供帮助,具有较好的应用价值和前景。
附图说明
图1:肺泡灌洗液外泌体分离流程图。
图2、肺泡灌洗液外泌体的TEM电镜图像。
图3、肺泡灌洗液外泌体NTA粒径分布图。
图4、外泌体标志蛋白Westernblot分析。
图5、ARDS肺泡灌洗液外泌体差异表达circRNA聚类分析。
图6、芯片筛选的差异表达显著的环状RNA的qPCR验证。
图7、hsa_circRNA_104034在正常人和ARDS患者肺泡灌洗液外泌体中的表达差异和受试者工作曲线。
图8、本发明与ARDS相关的血浆circRNA标志物检测方法的实验流程图。
图9、hsa_circRNA_104034在正常人和ARDS患者血浆中的表达差异(A)和受试者工作曲线(B)。
具体实施方式
实施例1
1.1患者样本收集
选取2020年5月至2021年5月期间在上海东方医院呼吸重症监护室(RICU)住院的重症肺炎致ARDS患者作为病例组。这些患者根据2012年发表的柏林定义诊断为ARDS,同时招募健康志愿者和诊断为肺部小结节的体检者作为对照组,年龄18-75岁。排除所有无法耐受支气管镜检查或存在支气管镜检查禁忌症的受试者。ARDS患者在诊断后48h内进行支气管镜肺泡灌洗。
1.2肺泡灌洗液收集
参照《肺部感染性疾病支气管肺泡灌洗病原体检测中国专家共识》(2017年版)及《支气管肺泡灌洗细胞学在间质性肺病的临床应用指南》(ATS 2012年版)要求采集支气管肺泡盥洗液(Bronchoalveolar Lavage Fluid,BALF)。所有患者排除禁忌后在静脉复合麻醉或2%利多卡因表面麻醉下行支气管镜检查,选择目标段支气管分次注入等量生理盐水后负压抽吸采集盥洗液20ml-80℃保存备用。
1.3肺泡灌洗液外泌体的分离
用超速离心法分离BALF外泌体。取BALF 20ml 500g离心5min,去除细胞。将上清液转移至新离心管2000g离心10分钟,10,000g离心30分钟,用0.22um滤膜过滤大囊泡,取1ml上清液100,000g离心70min取沉淀。PBS 100ul重悬外泌体。(肺泡灌洗液外泌体的分离流程见图1)
1.4外泌体的鉴定
使用Hitachi 7650透射电子显微镜(TEM)来鉴定分离的外泌体,应用纳米颗粒跟踪分析(NanoSight NS300)前述外泌体的粒径和浓度。用RIPA裂解肺泡灌洗液外泌体分离蛋白。BCA法对蛋白进行定量。SDS-PAGE跑胶,5%BSA封闭,转移至PVDF膜,一抗(CD9#13403,CD63 ab134045,CD86#91882)4℃孵育过夜。辣根过氧化物酶HRP标记的二抗37℃孵育1h。应用ECL试剂盒进行检测。IMAGE J软件进行图像分析。
1.5外泌体总RNA的提取
利用Trizol LS从肺泡灌洗液外泌体中抽提总RNA,用Nanodrop对RNA进行定量。
1.6CircRNA芯片高通量筛选
用RNase去除线性RNA,以富集环RNA。circRNAs经过扩增并转录成cy3标记的cRNA(Arraystar Super RNAlabeling Kit,Arraystar Inc.美国)。然后使用Arraystar HumancircRNA microarray进行杂交,并使用Agilent Scanner G2505C(Jamul,CA,USA)进行数据提取。
1.7逆转录反应
按照下述的逆转录反应体系进行逆转录反应
上述混合液在65℃水浴5min,冰上放置2min,短暂离心后,在下述的反应体系中继续反应。
按照上述反应体系混合后于37℃,恒温1分钟;移液枪轻轻吸打几次混合均匀,50℃温育60分钟,70℃温育15分钟使酶失活。将逆转录的cDNA置冰浴待用或-20℃保存。
1.8定量PCR扩增
定量PCR所使用的CircRNA引物序列见下表1:按照下述的反应体系实施定量PCR反应
将上述384-PCR板置于Realtime PCR仪上进行PCR反应。所有的指标均按以下程序进行:应用-ViiA7Real-time PCR System,95℃,10min;40个PCR循环(95℃,10秒;60℃,60秒,收集荧光)。为了建立PCR产物的熔解曲线,扩增反应结束后,按(95℃,10秒;60℃,60秒;95℃,15秒);并从60℃缓慢加热到99℃(仪器自动进行-Ramp Rate为0.05℃/秒)。
表1:定量PCR使用的CircRNA引物序列
2.9结果分析
使用公式2-△△CT计算circRNA表达的差异变化,student t-test计算pvalue,p值<0.05认为有统计学差异。
实验结果:
1、外泌体鉴定
透射电镜观察提取的外泌体:
TEM观察细胞外囊泡形态,外泌体呈茶盘状结构,与外泌体形态特征一致。(肺泡灌洗液外泌体TEM电镜图像见图2)
外泌体粒径分析:
NTA分析显示,分离的BALF外体的粒径分布在50至150nm之间(外泌体粒径分析结果详见图3)。
Western blot鉴定外泌体特异性分子标志物:
利用western blotting通过表达CD9和TSG101鉴定了这些细胞外囊泡的性质,结果证明这些细胞外囊泡是外泌体。随后,用抗CD86抗体检查了这些外来体的细胞来源,结果表明肺泡巨噬细胞可能是这些外来体的主要来源(Western blot鉴定外泌体特异性分子标志物结果详见图4)。
2、肺泡灌洗液外泌体circRNA芯片结果
circRNA芯片共检测到13,228个circRNA,其中629个circRNA在ARDS患者和健康受试者的BALF外体中差异表达(fold change>2,p<0.05),其中430个上调,199个下调。聚类热图均直观地显示了这些差异表达的circRNAs(circRNA芯片聚类图结果详见图5),红色表示高表达,绿色表示低表达。随后根据差异倍数>4、p值<0.005、原始信号值>1000等标准,筛选出前9位环状RNA进行RT-qPCR验证(芯片筛选的差异表达显著的环状RNA的qPCR验证结果详见图6),验证结果显示有7个circRNA指标与芯片结果相符。
3、肺泡灌洗液外泌体circ104034生物标志物的鉴定和检测
对筛选出在病例及对照组BALF外泌体中表达差异显著的环状RNA hsa_circRNA_104034进一步扩大样本量进行验证。
SEQ ID NO.1:CircRNA序列
>hsa_circRNA_104034
caagtcaagccagagatctgttatcaaaaatgttagtgattgatcctgacaagcggatctctgtagacgaagctctgcgtcacccatacatcactgtttggtatgaccccgccgaagcagaagccccaccacctcaaatttatgatgcccagttggaagaaagagaacatgcaattgaagaatggaaagagctaatttacaaagaagtcatggattgggaagaaagaagcaagaatggtgttgtaaaagatcagccttcag
应用RT-qPCR方法分别在ARDS病例组和对照组肺泡灌洗液外泌体中检测hsa_circRNA_104034的表达差异(ARDS患者的人口学特征详见表2),结果显示hsa_circRNA_104034在ARDS患者肺泡灌洗液外泌体中的表达明显高于正常组,ROC曲线下面积为1,诊断ARDS的敏感度是85%,特异度是90%(p<0.001)(hsa_circRNA_104034在正常人和ARDS患者肺泡灌洗液外泌体中的表达差异和受试者工作曲线结果详见图7)。
表2急性呼吸窘迫综合征(ARDS)患者的人口学和临床特征。
实施例2
2.1实验流程及样本收集
本发明与ARDS相关的血浆circRNA标志物检测方法的实验流程图详见图8。样本收集参考实施例1
2.2血浆的采集与制备
用EDTA抗凝管收集ARDS及正常人的全血3mL。采血后立即颠倒5次使得抗凝剂和血液充分混匀;血样采集后室温放置30min后,2000rpm离心10min,上清液即为血浆;保存于-80℃冰箱。
2.3血浆circRNA的提取
利用Invitrogen公司的Trizol LS从中抽提血浆总RNA,洗脱后-80℃保存备用。RNA的纯度和浓度用Nanodrop-1000进行检测。
2.4逆转录反应
参考实施例1中逆转录反应体系进行逆转录反应,上述混合液在65℃水浴5min,冰上放置2min,短暂离心后,按照实施例1的反应体系中继续反应。
按照上述反应体系混合后于37℃,恒温1分钟;移液枪轻轻吸打几次混合均匀,50℃温育60分钟,70℃温育15分钟使酶失活。将逆转录的cDNA置冰浴待用或-20℃保存。
2.5定量PCR扩增
参照实施例1中的反应体系完成定量PCR反应,将上述384-PCR板置于Real timePCR仪上进行PCR反应。所有的指标均按以下程序进行:应用-ViiA7Real-time PCR System,95℃,10min;40个PCR循环(95℃,10秒;60℃,60秒,收集荧光)。
为了建立PCR产物的熔解曲线,扩增反应结束后,按(95℃,10秒;60℃,60秒;95℃,15秒);并从60℃缓慢加热到99℃(仪器自动进行-Ramp Rate为0.05℃/秒)。定量PCR所使用的CircRNA引物序列参照实施例1中的引物学列进行反应,CircRNA序列>hsa_circRNA_104034同实施例1
结果分析:
使用公式2-△△CT计算circRNA表达的差异变化,student t-test计算pvalue,p值<0.05认为有统计学差异。
实验结果:
环状RNA circ104034生物标志物的鉴定和检测:在病例及对照组血浆中检测circ104034的表达差异,应用RT-qPCR方法分别在ARDS病例组和对照组血浆中检测circ104034的表达差异(ARDS患者的人口学特征详见实施例1),结果显示hsa_circRNA_104034在ARDS患者血浆中的表达明显高于正常组,ROC曲线下的面积是0.799,敏感性71.05%。特异性78.95%。(circ104034在正常人和ARDS患者血浆中的表达差异和受试者工作曲线结果详见图9,p<0.001)。
序列表
<110> 上海市东方医院(同济大学附属东方医院)
<120> 一种用于诊断急性呼吸窘迫综合征的circRNA标志物及检测试剂和试剂盒
<160> 23
<170> SIPOSequenceListing 1.0
<210> 1
<211> 261
<212> DNA
<213> Homo sapiens
<400> 1
caagtcaagc cagagatctg ttatcaaaaa tgttagtgat tgatcctgac aagcggatct 60
ctgtagacga agctctgcgt cacccataca tcactgtttg gtatgacccc gccgaagcag 120
aagccccacc acctcaaatt tatgatgccc agttggaaga aagagaacat gcaattgaag 180
aatggaaaga gctaatttac aaagaagtca tggattggga agaaagaagc aagaatggtg 240
ttgtaaaaga tcagccttca g 261
<210> 2
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
gtggccgagg actttgattg 20
<210> 3
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
cctgtaacaa cgcatctcat att 23
<210> 4
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
tgccctatgg gatgagaac 19
<210> 5
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
atggctggct cacttgtca 19
<210> 6
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
cttcacctca gcattacatt ca 22
<210> 7
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
gtcaaggact ggagacctca a 21
<210> 8
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
aaagaagtca tggattggga a 21
<210> 9
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
cttgtcagga tcaatcacta aca 23
<210> 10
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
ccagaattag aaaaagaaag cc 22
<210> 11
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
aacttgctga tttccctgta ga 22
<210> 12
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
gacggtggtc acgcctgtt 19
<210> 13
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
cctctgctga aagcttggga 20
<210> 14
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
cattccagtt ttcctgatgg t 21
<210> 15
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
attactcttc ttatttgtgg cttc 24
<210> 16
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
ctgcacgtct tcaccttcg 19
<210> 17
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
ctgctgcttc ccgttcttac 20
<210> 18
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
cagacctacc ttcagtcaac aa 22
<210> 19
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
gtgtgtgtag ccatattttt cat 23
<210> 20
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
aatacgaatg gcaccgcttc 20
<210> 21
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
agtagtgagg ccgcttataa cc 22
<210> 22
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
tttcaatgat gatgctatgc tg 22
<210> 23
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
ttatggattt ggatgtgctc g 21
Claims (7)
1.一种circRNA,其特征在于,其核苷酸序列如SEQ ID NO.1所示。
2.一种用于诊断急性呼吸窘迫综合征的circRNA标志物,其特征在于,所述circRNA标志物为hsa_circRNA_104034,所述的hsa_circRNA_104034核苷酸序列如SEQ ID NO.1所示。
3.circRNA标志物hsa_circRNA_104034在制备作为急性呼吸窘迫综合征检测标志物中的应用,所述的hsa_circRNA_104034核苷酸序列如SEQ ID NO.1所示。
4.检测circRNA标志物hsa_circRNA_104034的试剂在制备用于诊断急性呼吸窘迫综合征的试剂盒中的应用,所述的hsa_circRNA_104034核苷酸序列如SEQ ID NO.1所示。
5.根据权利要求4所述的应用,其特征在于,所述的试剂包括SEQ ID NO.2~23所示的引物。
6.一种试剂盒,其特征在于,包括检测circRNA标志物hsa_circRNA_104034的试剂,所述的hsa_circRNA_104034的核苷酸序列如SEQ ID NO.1所示。
7.根据权利要求5所述的一种试剂盒,其特征在于,所述的试剂包括SEQ ID NO.2~23所示的引物。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210770010.8A CN115011602B (zh) | 2022-06-30 | 2022-06-30 | 一种用于诊断急性呼吸窘迫综合征的circRNA标志物及检测试剂和试剂盒 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210770010.8A CN115011602B (zh) | 2022-06-30 | 2022-06-30 | 一种用于诊断急性呼吸窘迫综合征的circRNA标志物及检测试剂和试剂盒 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN115011602A true CN115011602A (zh) | 2022-09-06 |
CN115011602B CN115011602B (zh) | 2024-01-26 |
Family
ID=83078358
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202210770010.8A Active CN115011602B (zh) | 2022-06-30 | 2022-06-30 | 一种用于诊断急性呼吸窘迫综合征的circRNA标志物及检测试剂和试剂盒 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN115011602B (zh) |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1661110A (zh) * | 2003-12-22 | 2005-08-31 | 霍夫曼-拉罗奇有限公司 | 来自脂肪组织的肥胖症新靶 |
US20180023079A1 (en) * | 2015-02-03 | 2018-01-25 | Johann Wolfgang Goethe-Universität Frankfurt am Main | Circular RNA For The Diagnosis Of Cardiovascular And Inflammatory Diseases |
-
2022
- 2022-06-30 CN CN202210770010.8A patent/CN115011602B/zh active Active
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1661110A (zh) * | 2003-12-22 | 2005-08-31 | 霍夫曼-拉罗奇有限公司 | 来自脂肪组织的肥胖症新靶 |
US20180023079A1 (en) * | 2015-02-03 | 2018-01-25 | Johann Wolfgang Goethe-Universität Frankfurt am Main | Circular RNA For The Diagnosis Of Cardiovascular And Inflammatory Diseases |
Non-Patent Citations (1)
Title |
---|
SHIQING SHAO: "Hsa_circ_0075341 is up-regulated and exerts oncogenic properties by sponging miR-149-5p in cervical cancer", BIOMEDICINE & PHARMACOTHERAPY, vol. 2020, no. 121, pages 1 - 7 * |
Also Published As
Publication number | Publication date |
---|---|
CN115011602B (zh) | 2024-01-26 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN109097477B (zh) | 一种用于乳腺癌诊断的circRNA标志物及其应用 | |
CN109837343B (zh) | 早期肺腺癌特异性外泌体miRNA及其应用 | |
Han et al. | Tumor-derived circulating exosomal miR-342-5p and miR-574-5p as promising diagnostic biomarkers for early-stage Lung Adenocarcinoma | |
CN111172287B (zh) | 外泌体lncRNA RN7SL5P作为内参基因在胃癌lncRNA检测中的应用 | |
CN108070641B (zh) | 基于qPCR或数字PCR技术用于检测囊泡中AR-V7及AR的引物及探针 | |
CN109868315B (zh) | 用于早期检测脑动脉瘤性蛛网膜下腔出血严重程度及预后的体外方法 | |
CN109609630B (zh) | 用于早期胃癌诊断的分子标志物及其应用 | |
CN107881239B (zh) | 血浆中与结直肠癌转移相关的miRNA标志物及其应用 | |
CN113999909B (zh) | 一种用于胃癌诊断的血清外泌体标志物及其应用、扩增引物对和诊断试剂盒 | |
JP2023113877A (ja) | 膵がんの検出を補助する方法 | |
CN113337613B (zh) | 一种与肝癌相关的血清外泌体tsRNA标志物、探针及其应用 | |
CN108034722B (zh) | 一种lncRNA SGOL1-AS1在作为胃癌诊断标志物的应用 | |
WO2019117257A1 (ja) | 乳がんの検出を補助する方法 | |
CN106939354B (zh) | miRNA-4530作为肺癌诊断标志物的应用 | |
CN115011602B (zh) | 一种用于诊断急性呼吸窘迫综合征的circRNA标志物及检测试剂和试剂盒 | |
CN107904310B (zh) | 用于大肠癌诊断的尿液microRNA生物标志物、试剂盒及其应用 | |
CN114958859B (zh) | 一种用于诊断急性呼吸窘迫综合征的circRNA标志物及诊断试剂 | |
JPWO2019117270A1 (ja) | 頭頸部がんの検出を補助する方法 | |
CN111455037B (zh) | 基于血浆外泌体环状rna的冠心病分子诊断标志物及其应用 | |
CN106929599B (zh) | miRNA-6126作为肺癌诊断标志物的应用 | |
JP6441567B2 (ja) | 小胞内のポリヌクレオチドを含む乳がん診断用組成物及びキット、並びにそれを利用した乳がん診断方法 | |
WO2019095541A1 (zh) | 一种诊断预示乳腺癌骨转移的组合物及诊断预示方法 | |
CN110468204A (zh) | 生物标志物及其在制备诊断ptc的产品中的应用 | |
CN112176060B (zh) | 一组血浆非编码rna及检测其表达水平的引物组与结直肠癌检测试剂盒 | |
CN110257514A (zh) | 一种新的食管癌血液miRNA标志物及其应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |