CN114214404A - 外泌体microRNA和mRNA的应用及其试剂盒 - Google Patents
外泌体microRNA和mRNA的应用及其试剂盒 Download PDFInfo
- Publication number
- CN114214404A CN114214404A CN202210041734.9A CN202210041734A CN114214404A CN 114214404 A CN114214404 A CN 114214404A CN 202210041734 A CN202210041734 A CN 202210041734A CN 114214404 A CN114214404 A CN 114214404A
- Authority
- CN
- China
- Prior art keywords
- mir
- kif5c
- exosome
- disease
- primer
- 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.)
- Pending
Links
- 210000001808 exosome Anatomy 0.000 title claims abstract description 50
- 108020004999 messenger RNA Proteins 0.000 title claims abstract description 14
- 108700011259 MicroRNAs Proteins 0.000 title abstract description 16
- 239000002679 microRNA Substances 0.000 title abstract description 16
- 208000018737 Parkinson disease Diseases 0.000 claims abstract description 32
- 108091071567 miR-1307 stem-loop Proteins 0.000 claims abstract description 30
- 210000002700 urine Anatomy 0.000 claims abstract description 19
- 238000012216 screening Methods 0.000 claims abstract description 9
- 101001006789 Homo sapiens Kinesin heavy chain isoform 5C Proteins 0.000 claims abstract 9
- 102100027928 Kinesin heavy chain isoform 5C Human genes 0.000 claims abstract 9
- 238000010839 reverse transcription Methods 0.000 claims description 14
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 11
- 238000003753 real-time PCR Methods 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- 239000003147 molecular marker Substances 0.000 claims description 9
- 238000001514 detection method Methods 0.000 claims description 8
- 239000000463 material Substances 0.000 claims description 8
- 239000003153 chemical reaction reagent Substances 0.000 claims description 7
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 claims description 6
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 6
- 230000003321 amplification Effects 0.000 claims description 6
- 238000000605 extraction Methods 0.000 claims description 6
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 claims description 6
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 5
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 4
- 102100034343 Integrase Human genes 0.000 claims description 4
- 238000002123 RNA extraction Methods 0.000 claims description 4
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 claims description 4
- 239000008367 deionised water Substances 0.000 claims description 4
- 229910021641 deionized water Inorganic materials 0.000 claims description 4
- 239000003161 ribonuclease inhibitor Substances 0.000 claims description 4
- 229960001701 chloroform Drugs 0.000 claims description 3
- 239000006166 lysate Substances 0.000 claims description 3
- 239000012621 metal-organic framework Substances 0.000 claims description 3
- 238000010606 normalization Methods 0.000 claims description 3
- 239000007853 buffer solution Substances 0.000 claims description 2
- 239000002299 complementary DNA Substances 0.000 claims description 2
- 239000012154 double-distilled water Substances 0.000 claims description 2
- 239000011259 mixed solution Substances 0.000 claims description 2
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 2
- 239000012498 ultrapure water Substances 0.000 claims description 2
- 238000003745 diagnosis Methods 0.000 abstract description 4
- 238000012360 testing method Methods 0.000 abstract description 2
- 238000012795 verification Methods 0.000 abstract 1
- 239000006228 supernatant Substances 0.000 description 11
- 238000000034 method Methods 0.000 description 8
- 108090000623 proteins and genes Proteins 0.000 description 8
- 201000010099 disease Diseases 0.000 description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 239000000090 biomarker Substances 0.000 description 3
- 238000007664 blowing Methods 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 235000018102 proteins Nutrition 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 208000024891 symptom Diseases 0.000 description 3
- 102000052052 Casein Kinase II Human genes 0.000 description 2
- 108010010919 Casein Kinase II Proteins 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 210000001124 body fluid Anatomy 0.000 description 2
- 239000010839 body fluid Substances 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- SLPJGDQJLTYWCI-UHFFFAOYSA-N dimethyl-(4,5,6,7-tetrabromo-1h-benzoimidazol-2-yl)-amine Chemical compound BrC1=C(Br)C(Br)=C2NC(N(C)C)=NC2=C1Br SLPJGDQJLTYWCI-UHFFFAOYSA-N 0.000 description 2
- 210000002487 multivesicular body Anatomy 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 108091032955 Bacterial small RNA Proteins 0.000 description 1
- 206010006100 Bradykinesia Diseases 0.000 description 1
- 206010048409 Brain malformation Diseases 0.000 description 1
- 108090000994 Catalytic RNA Proteins 0.000 description 1
- 102000053642 Catalytic RNA Human genes 0.000 description 1
- 206010010774 Constipation Diseases 0.000 description 1
- 206010070666 Cortical dysplasia Diseases 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 206010017577 Gait disturbance Diseases 0.000 description 1
- 208000006083 Hypokinesia Diseases 0.000 description 1
- 102000010638 Kinesin Human genes 0.000 description 1
- 108010063296 Kinesin Proteins 0.000 description 1
- WTDRDQBEARUVNC-LURJTMIESA-N L-DOPA Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C(O)=C1 WTDRDQBEARUVNC-LURJTMIESA-N 0.000 description 1
- WTDRDQBEARUVNC-UHFFFAOYSA-N L-Dopa Natural products OC(=O)C(N)CC1=CC=C(O)C(O)=C1 WTDRDQBEARUVNC-UHFFFAOYSA-N 0.000 description 1
- 208000000676 Malformations of Cortical Development Diseases 0.000 description 1
- 208000002740 Muscle Rigidity Diseases 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 206010071390 Resting tremor Diseases 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 208000032023 Signs and Symptoms Diseases 0.000 description 1
- 206010044565 Tremor Diseases 0.000 description 1
- 108091023045 Untranslated Region Proteins 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 238000010009 beating Methods 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 208000023397 cerebral cortical dysplasia Diseases 0.000 description 1
- 208000012056 cerebral malformation Diseases 0.000 description 1
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000002651 drug therapy Methods 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 239000003517 fume Substances 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 229960004502 levodopa Drugs 0.000 description 1
- 210000003712 lysosome Anatomy 0.000 description 1
- 230000001868 lysosomic effect Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 108091070501 miRNA Proteins 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 210000000653 nervous system Anatomy 0.000 description 1
- 208000015122 neurodegenerative disease Diseases 0.000 description 1
- 108091027963 non-coding RNA Proteins 0.000 description 1
- 102000042567 non-coding RNA Human genes 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 238000000643 oven drying Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 230000001144 postural effect Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 210000001995 reticulocyte Anatomy 0.000 description 1
- 108091092562 ribozyme Proteins 0.000 description 1
- 210000003296 saliva Anatomy 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 208000019116 sleep disease Diseases 0.000 description 1
- 208000022925 sleep disturbance Diseases 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 210000004895 subcellular structure Anatomy 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
Images
Classifications
-
- 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
- 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
- C12Q1/6851—Quantitative amplification
-
- 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
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明涉及生物技术领域，尤其涉及外泌体microRNA和mRNA的应用及其试剂盒。本发明通过试验证明了尿液外泌体miR‑1307‑3p和KIF5C在正常人和帕金森氏病患者中表达存在显著差异，说明其可以成为筛查和/或诊断帕金森氏病的分子标志物，两种分子标志物组合可以准确地区分帕金森氏病患者和健康人，实现帕金森氏病的早期筛查和诊断。
Description
技术领域
本发明涉及生物技术领域，尤其涉及外泌体microRNA和mRNA的应用及其试剂盒。
背景技术
帕金森氏病(Parkinson’s disease，PD)是一种常见的神经系统变性疾病，老年人多见，平均发病年龄为60岁左右，40岁以下起病的青年帕金森氏病较少见。帕金森氏病的临床表现主要包括静止性震颤、运动迟缓、肌强直和姿势步态障碍，同时患者可伴有抑郁、便秘和睡眠障碍等非运动症状。帕金森氏病的诊断主要依靠病史、临床症状及体征。一般的辅助检查多无异常改变。药物治疗是帕金森氏病最主要的治疗手段，左旋多巴制剂仍是最有效的药物。手术治疗是药物治疗的一种有效补充。康复治疗、心理治疗及良好的护理也能在一定程度上改善症状。目前应用的治疗手段虽然只能改善症状，不能阻止病情的进展，也无法治愈疾病。因此，帕金森氏病的早期诊断和早期治疗有助于延缓病情进展，提高患者生存质量。
外泌体(exosome)是一些小的、由细胞分泌且有膜包裹的亚细胞结构，包含有RNA和蛋白质，其直径通常为30-150nm。现今，外泌体特指直径在40-100nm的盘状囊泡。1983年，外泌体首次于绵羊网织红细胞中被发现，1987年Johnstone将其命名为“exosome”。多种细胞在正常及病理状态下均可分泌外泌体。其主要来源于细胞内溶酶体微粒内陷形成的多囊泡体，经多囊泡体外膜与细胞膜融合后释放到胞外基质中。外泌体天然存在于体液中，包括血液、唾液、尿液、脑脊液和乳汁中。近年来，基于外泌体寻找特定疾病的相关生物标志物逐渐引起研究人员的关注。特别是2007年发现外泌体含有丰富的小RNA，尤其是microRNA之后，利用外泌体microRNA筛查疾病的相关分子标志物得到了长足的发展。MicroRNA是一些小的非编码RNA，长度在21-25个碱基。microRNA靶向针对信使RNA3’非翻译区来抑制相关基因翻译。即使大量核酶存在，microRNA也依然稳定存在于各类体液中，因而被广泛用作生物标志物。
KIF5C编码的蛋白是驱动蛋白的一条重链的亚基，参与中枢神经系统内的物质传输。该蛋白通过与另一条重链和两条轻链结合而作为四聚体与酪蛋白激酶II(CK2)相互作用。该基因的突变与皮质发育不良合并其他脑畸形有关。
发明内容
有鉴于此，本发明目的在于提供外泌体miR-1307-3p和/或KIF5C作为分子标志物在制备筛查和/或诊断帕金森氏病的产品中的应用，以及检测外泌体miR-1307-3p和/或KIF5C的试剂在制备筛查和/或诊断帕金森氏病的产品中的应用。
外泌体miR-1307-3p为microRNA分子标志物，其核苷酸序列为ACUCGGCGUGGCGUCGGUCGUG(SEQ ID NO.1)。
KIF5C为mRNA分子标志物，全称是KinesinFamilyMember 5C，为KIF5C的mRNA全长，长度为6954bp。
本发明中，所述外泌体为尿液外泌体；所述产品为试剂盒；所述试剂盒优选为实时荧光定量PCR检测试剂盒。
本发明还提供了一种筛查和/或诊断帕金森氏病的实时荧光定量PCR检测试剂盒，包括尿液外泌体提取系统，外泌体RNA提取系统，反转录系统，扩增外泌体miR-1307-3p和/或KIF5C的系统及其相对定量内参标准化系统。
进一步地，所述miR-1307-3p的序列的相对定量内参标准化系统由U6组成；
所述KIF5C的相对定量内参标准化系统由GAPDH组成；
所述尿液外泌体提取系统包括金属有机骨架材料uio-66；
所述外泌体总RNA提取系统包括RNAiso Plus裂解液、三氯甲烷、异丙醇、RNase-free ddH2O(无酶水)；
所述反转录扩增系统包括RNA模板、Random/Oligo(dT)引物、miR-1307-3p引物、U6引物、dNTP Mix(dNTP混合物)、反转录酶、5×反转录缓冲液、RNA酶抑制剂、无RNA酶的去离子水；
所述荧光定量PCR扩增系统包括反转录扩增得到的cDNA模板、SYBR Green混合液、miR-1307-3p引物、KIF5C引物、超纯水。
本发明所提供的外泌体microRNA分子标志物是miR-1307-3p，mRNA分子标志物是KIF5C，上述两种分子标志物联合用于诊断帕金森氏病，能够更准确更及时地对帕金森氏病进行筛查和诊断。本发明提供的试剂盒通过定量检测尿液外泌体microRNA分子标志物miR-1307-3p和mRNA分子标志物KIF5C来筛查和诊断帕金森氏病。由于尿液具有取样方便、没有创伤性且可进行连续检测的优点，因此从尿液中寻找生物标志物可以将帕金森氏病的早期筛查和诊断提高至一个新的水平；同时，通过实时荧光定量PCR技术对帕金森氏病患者尿液外泌体microRNA和mRNA进行检测，定量准确。
附图说明
图1是用U6进行相对定量内参标准化后健康志愿者和诊断为PD患者尿液外泌体中miR-1307-3p的表达情况；
图2是用GAPDH进行相对定量内参标准化健康志愿者和诊断为PD患者尿液外泌体中KIF5C的表达情况；
图3是用miR-1307-3p的RQ值诊断PD患者的ROC分析结果；
图4是用KIF5C的RQ值诊断PD患者的ROC分析结果；
图5是用miR-1307-3p和KIF5C的RQ值联合诊断PD患者的ROC分析结果。
具体实施方式
本发明提供了外泌体microRNA和mRNA的应用及其试剂盒。本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
如无特殊说明，本发明采用的试材皆为普通市售品，皆可于市场购得。
下面结合实施例，进一步阐述本发明：
实施例1检测帕金森氏病病人尿液外泌体样本、健康对照受试者尿液外泌体样本中miR-1307和KIF5C的表达
1.试剂与材料
本发明中使用初次诊断为PD的病人尿液样本29例和健康对照受试者样本20例。
实验试剂主要有金属有机骨架材料uio-66-DSPE、RNA iso plus、Random/Oligo(dT)引物、miR-1307-3p引物、U6引物、KIF5C引物、GAPDH引物、dNTPMix(dNTP混合物)、反转录酶、5×反转录缓冲液、RNA酶抑制剂、无RNA酶的去离子水等。
2.实验过程：
(1)尿液样本的处理
取尿液样本，2700转，4℃离心30分钟，分离沉淀和上清。取1mL上清至1.5mLEP管中。
(2)外泌体的提取
加入100μL 2mg/mLuio 66-DSPE-COOH材料、100μLPBS溶液和100μL0.1％TritonX-100和NP-40溶液，混匀后360°摇床反应1小时。然后，5000转，室温离心5分钟；吸去上清，加入1mL 0.01％TritonX-100和NP-40溶液，将底部沉淀吹匀后，5000转，室温离心5分钟；吸去上清，加入1mLPBS溶液，将底部沉淀吹匀后，5000转，室温离心5分钟；吸去上清。
(3)外泌体总RNA的提取
每个EP管中加入500μL RNAiso Plus裂解液，室温静置15min，使充分裂解；4℃，12000g，离心5min后，加入100μL三氯甲烷，盖紧盖子，上下翻转10下使其混匀，室温静置5min；4℃，12000g，离心15min，RNA存在于上层水相中。吸取200μL上清于RNase-free EP管中，加入与上清等体积的200μL异丙醇，上下翻转10下使其混匀，室温静置10min；4℃，12000g，离心10min，RNA沉淀于管底。弃去上清，倒置于纸巾上，吸干上清，去除有机溶剂；向每个EP管中加入1mL 75％乙醇(DEPC水配制)，4℃，12000g，离心5min；弃去上清，倒置在纸巾上，吸干水分，置于通风橱内烘干20min；加入20μL经灭菌处理的DEPC水溶解RNA沉淀，枪尖轻轻吹打混匀，Nanodrop紫外分光光度计测定其浓度和纯度，置-80℃保存。
(4)RNA的逆转录
由于microRNA特异性，U6和miR-1307-3p使用特定引物进行单独逆转录与扩增，GAPDH和KIF5C使用Random/Oligo(dT)进行逆转录与扩增，所需试剂如下表所示：
表1
| 材料 | 体系 |
| RNA模板 | 1000ng |
| U6反转录引物 | 1μL |
| miR-1307-3p反转录引物 | 1μL |
| Random/Oligo(dT) | 2μL |
| DEPC水 | 至11μL |
| 5×反转录缓冲液 | 5μL |
| dNTP(10mM) | 2μL |
| 反转录酶(200U/μL) | 0.5μL |
| RNA酶抑制剂(40U/μL) | 0.5μL |
| DEPC水 | 6μL |
| 总体积 | 25μL |
逆转录程序为：第一次补足DEPC水至11μL后，70℃反应10min后冰浴2min，后加余下反应试剂至补足DEPC水至25μL，42℃反应60min，70℃反应10min。
(5)实时荧光定量PCR
所需试剂如下表：
表2
| 材料 | 体系(μL) |
| 反转录产物 | 2 |
| 正向引物(10μM) | 1 |
| 反向引物(10μM) | 1 |
| SYBRGreen | 10 |
| 灭菌去离子水 | 6 |
| 总体积 | 20 |
采用如下程序进行实时荧光定量PCR反应：
表3
(6)数据处理
用SYBR Green作为荧光染料，以U6作为miR-1307-3p的内参基因，以GAPDH作为KIF5C的内参基因。目的基因的相对表达率(RQ)采用Ct方法计算:RQ＝2-△△Ct(Ct表示反应的实时荧光强度显著大于背景值时的循环数；△Ctsample＝CtsampleCtU6sample,△Ctcontrol＝Ctcontrol-CtU6control,△△Ct＝△Ctsample-△Ctcontrol；△Ctsample＝CtsampleCtGAPDHsample,△Ctcontrol＝Ctcontrol-CtGAPDHcontrol,△△Ct＝△Ctsample-△Ctcontrol)。结果见图1～2。
(7)结果判断
ROC分析结果如图3和图4所示，当miR-1307-3p的RQ值≥1.5(AUC＝0.880；P＝3.91×10-7)时，受试者为帕金森氏病病人；当KIF5C的RQ值≤1.35(AUC＝0.850；P＝2.86×10-6)时，受试者为帕金森氏病病人。
如图5所示，当用miR-1307-3p的RQ值与KIF5C的RQ值两个指标进行联合诊断时，按照公式PRE＝-0.665-1.203×RQ(KIF5C)+1.018×RQ(miR-1307-3p)。当PRE值≥0.53(AUC＝0.902；P＝8.14×10-8)时，受试者为帕金森氏病病人。
以上仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 苏州大学
<120> 外泌体microRNA和mRNA的应用及其试剂盒
<130> MP21026940
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 1
acucggcgug gcgucggucg ug 22
Claims (10)
1.外泌体miR-1307-3p和/或KIF5C mRNA作为分子标志物在制备筛查和/或诊断帕金森氏病的产品中的应用。
2.检测外泌体miR-1307-3p和/或KIF5C的试剂在制备筛查和/或诊断帕金森氏病的产品中的应用。
3.根据权利要求1或2所述的应用，其特征在于，所述miR-1307-3p的序列如SEQ IDNO.1所示。
4.根据权利要求1或2所述的应用，其特征在于，所述KIF5C为全长序列。
5.根据权利要求1或2所述的应用，其特征在于，所述外泌体为尿液外泌体。
6.根据权利要求1或2任一项所述的应用，其特征在于，所述产品为试剂盒。
7.根据权利要求6所述的应用，其特征在于，所述试剂盒为实时荧光定量PCR检测试剂盒。
8.根据权利要求7所述的应用，其特征在于，所述实时荧光定量PCR检测试剂盒包括扩增miR-1307-3p和/或KIF5C mRNA的引物。
9.一种筛查和/或诊断帕金森氏病的实时荧光定量PCR检测试剂盒，其特征在于，包括扩增外泌体miR-1307-3p和/或KIF5C的系统及其相对定量内参标准化系统、尿液外泌体提取系统、外泌体RNA提取系统和反转录系统。
10.根据权利要求9所述的实时荧光定量PCR检测试剂盒，其特征在于，所述miR-1307-3p的序列的相对定量内参标准化系统由U6组成；
所述KIF5C的相对定量内参标准化系统由GAPDH组成；
所述尿液外泌体提取系统包括金属有机骨架材料uio-66；
所述外泌体总RNA提取系统包括RNAiso Plus裂解液、三氯甲烷、异丙醇、RNase-freeddH2O；
所述反转录扩增系统包括RNA模板、Random/Oligo(dT)引物、miR-1307-3p引物、U6引物、dNTPMix、反转录酶、5×反转录缓冲液、RNA酶抑制剂、无RNA酶的去离子水；
所述荧光定量PCR扩增系统包括反转录扩增得到的cDNA模板、SYBR Green混合液、miR-1307-3p引物、KIF5C引物、超纯水。
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210041734.9A CN114214404A (zh) | 2022-01-14 | 2022-01-14 | 外泌体microRNA和mRNA的应用及其试剂盒 |
| PCT/CN2022/086214 WO2023134037A1 (zh) | 2022-01-14 | 2022-04-12 | 外泌体microRNA和mRNA的应用及其试剂盒 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210041734.9A CN114214404A (zh) | 2022-01-14 | 2022-01-14 | 外泌体microRNA和mRNA的应用及其试剂盒 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114214404A true CN114214404A (zh) | 2022-03-22 |
Family
ID=80708242
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210041734.9A Pending CN114214404A (zh) | 2022-01-14 | 2022-01-14 | 外泌体microRNA和mRNA的应用及其试剂盒 |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN114214404A (zh) |
| WO (1) | WO2023134037A1 (zh) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023134037A1 (zh) * | 2022-01-14 | 2023-07-20 | 苏州大学 | 外泌体microRNA和mRNA的应用及其试剂盒 |
| WO2024098369A1 (zh) * | 2022-11-11 | 2024-05-16 | 北京市神经外科研究所 | 一种基于dna六面体的帕金森病体外诊断试剂盒及其应用 |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105441578A (zh) * | 2016-01-19 | 2016-03-30 | 中南大学湘雅二医院 | 尿液外泌体microRNA分子标志物的应用及制备的试剂盒 |
| CN110592207A (zh) * | 2019-09-23 | 2019-12-20 | 中南大学湘雅二医院 | 一种外泌体microRNA的应用及制备的试剂盒 |
| CN111748618A (zh) * | 2020-07-27 | 2020-10-09 | 河南科技大学第一附属医院 | 一种帕金森病早期诊断的生物标志物及其应用 |
| CN111944809A (zh) * | 2020-07-28 | 2020-11-17 | 武汉睿健医药科技有限公司 | 帕金森病的诊断标志物及其应用 |
| KR20210019232A (ko) * | 2019-08-12 | 2021-02-22 | 아주대학교산학협력단 | 조기 간암 진단을 위한 엑소좀 유래 microRNA 바이오마커 및 그 용도 |
| CN112575079A (zh) * | 2020-12-31 | 2021-03-30 | 郑州大学第一附属医院 | 外泌体miRNA作为诊断2型糖尿病合并冠心病的分子标志物及其应用 |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013036936A1 (en) * | 2011-09-09 | 2013-03-14 | Van Andel Research Institute | Microrna biomarkers for diagnosing parkinson's disease |
| GB201302468D0 (en) * | 2013-02-12 | 2013-03-27 | Reneuron Ltd | Stem cell product |
| WO2016196822A1 (en) * | 2015-06-02 | 2016-12-08 | Cedars-Sinai Medical Center | Urodele exosomes as therapeutic agents |
| CN113755597B (zh) * | 2021-10-14 | 2022-12-06 | 杭州师范大学 | 外周血外泌体miRNA联合标志物在制备检测HBV阳性肝硬化早期肝癌试剂盒中的应用 |
| CN114214404A (zh) * | 2022-01-14 | 2022-03-22 | 苏州大学 | 外泌体microRNA和mRNA的应用及其试剂盒 |
-
2022
- 2022-01-14 CN CN202210041734.9A patent/CN114214404A/zh active Pending
- 2022-04-12 WO PCT/CN2022/086214 patent/WO2023134037A1/zh unknown
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105441578A (zh) * | 2016-01-19 | 2016-03-30 | 中南大学湘雅二医院 | 尿液外泌体microRNA分子标志物的应用及制备的试剂盒 |
| KR20210019232A (ko) * | 2019-08-12 | 2021-02-22 | 아주대학교산학협력단 | 조기 간암 진단을 위한 엑소좀 유래 microRNA 바이오마커 및 그 용도 |
| CN110592207A (zh) * | 2019-09-23 | 2019-12-20 | 中南大学湘雅二医院 | 一种外泌体microRNA的应用及制备的试剂盒 |
| CN111748618A (zh) * | 2020-07-27 | 2020-10-09 | 河南科技大学第一附属医院 | 一种帕金森病早期诊断的生物标志物及其应用 |
| CN111944809A (zh) * | 2020-07-28 | 2020-11-17 | 武汉睿健医药科技有限公司 | 帕金森病的诊断标志物及其应用 |
| CN112575079A (zh) * | 2020-12-31 | 2021-03-30 | 郑州大学第一附属医院 | 外泌体miRNA作为诊断2型糖尿病合并冠心病的分子标志物及其应用 |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023134037A1 (zh) * | 2022-01-14 | 2023-07-20 | 苏州大学 | 外泌体microRNA和mRNA的应用及其试剂盒 |
| WO2024098369A1 (zh) * | 2022-11-11 | 2024-05-16 | 北京市神经外科研究所 | 一种基于dna六面体的帕金森病体外诊断试剂盒及其应用 |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2023134037A1 (zh) | 2023-07-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN114214404A (zh) | 外泌体microRNA和mRNA的应用及其试剂盒 | |
| Chen et al. | Increased salivary microRNAs that regulate DJ-1 gene expression as potential markers for Parkinson’s disease | |
| WO2008127317A2 (en) | Alternatively spliced pre-mrna transcripts as biomarkers in idiopathic neurodegenerative diseases | |
| US20180305754A1 (en) | Biomarker for diagnosis of drug addiction and kit for diagnosis of drug addiction | |
| CN104928373A (zh) | 一种帕金森病的基因诊断试剂盒 | |
| CN106434982A (zh) | 缺血性脑卒中相关的分子标记物及其应用 | |
| AU2016351311B9 (en) | SCAP gene mutant and the application thereof | |
| CN110551728B (zh) | Foxc1基因突变体及其应用 | |
| KR101939368B1 (ko) | 알츠하이머성 치매 진단용 바이오마커 및 이의 용도 | |
| CN107460241B (zh) | 外泌体小分子rna在急性心肌梗死风险评估中的应用 | |
| CN108004313A (zh) | 早发冠心病致病基因及其体外检测的试剂、制剂或试剂盒和应用 | |
| JP2012019784A (ja) | 疲労の判定方法 | |
| CN113774127B (zh) | 血清细胞外囊泡miR-503-5p在制备2型糖尿病发病的诊断试剂盒中的应用 | |
| EP4370712A1 (en) | Methods of detecting sjögren's syndrome using salivary exosomes | |
| CN109355373B (zh) | circRNA在制备增生性玻璃体视网膜病变诊断试剂中的应用 | |
| CN1316038C (zh) | 检测与人类遗传性耳聋相关的wfs1基因突变的试剂盒 | |
| CN111349698A (zh) | 与中枢神经系统感染疾病相关的外泌体内核酸标志物及其应用 | |
| CN110283902A (zh) | 一种肺动脉高压环状rna分子标志物、其制备方法和应用试剂盒 | |
| Leslie et al. | Pompe Disease Synonyms: Acid Alpha-Glucosidase Deficiency, Acid Maltase Deficiency, GAA Deficiency, Glycogen Storage Disease Type II (GSD II), Glycogenosis Type II | |
| CN116694761B (zh) | 用于诊断乳腺癌化疗心脏损伤的piRNA标志物、试剂盒及其应用 | |
| KR102555467B1 (ko) | 과민성 대장 증후군의 진단 또는 예측용 엑소좀 바이오마커 및 이의 용도 | |
| WO2022253196A1 (zh) | Synaptotagmin-11 mRNA作为帕金森综合征标志物及其用途 | |
| RU2283494C1 (ru) | Способ прогнозирования развития токсического гепатита при химиотерапии множественной миеломы | |
| CN114350793A (zh) | 一种外泌体miRNA在制备慢性疼痛-抑郁共病诊断产品中的应用 | |
| CN115873939A (zh) | 用于帕金森病诊断、辅助诊断或早期筛查的引物组 |
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 |