Exosome RNA molecular marker combination for diagnosis of colorectal adenoma and application thereof
A molecular marker and colorectal technology, applied in the direction of DNA / RNA fragments, recombinant DNA technology, microbial measurement / inspection, etc., can solve the problems of bleeding, perforation, lack of sensitivity and specificity, etc.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Publication Date
- 2020-07-28
Smart Images
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Abstract
Description
technical field
[0001] The present invention relates to the field of biological detection, more specifically to biological molecular detection, and more specifically to a group of exosome RNA markers related to colorectal adenoma and applications thereof. Background technique
[0002] Colorectal adenoma is a benign tumor originating from the glandular epithelium of the colorectal mucosa, but it is closely related to the occurrence of colorectal cancer. Studies have shown that about 80% of colorectal cancers develop from colorectal adenoma, so colorectal adenoma is Considered a precancerous lesion of colorectal cancer. As the incidence and mortality of colorectal cancer in my country are increasing year by year, studies have shown that timely detection and removal of colorectal adenomas can reduce the incidence of colorectal cancer by 76%. Rectal carcinogenesis is critical.
[0003] Electronic colonoscopy is helpful to discover and observe colorectal mucosal lesions, combine...
Examples
Embodiment 1
[0041] Example 1 Screening of exosomal RNA molecular markers associated with colorectal adenoma based on high-throughput sequencing
[0042]No less than 10 mL of blood was collected from 15 patients with early colorectal adenoma and 15 healthy subjects in the control group. a comparison with plasma total miRNAs, L Min, S Zhu, L Chen, X Liu, R Wei, L Zhao, Y Yang, Z Zhang, GKong, Journal of extracellular vesicles 8(1), 1643670) isolated exosomes in plasma , QIAGEN miRNeasy mini kit was used to extract RNA, and the prepared RNA was subjected to micro strand-specific RNA library construction and sequencing (Evaluation of circulating small extracellular vesicles derived miRNAs asbiomarkers of early colon cancer: a comparison with plasma total miRNAs, L Min, S Zhu , L Chen, X Liu, R Wei, L Zhao, Y Yang, Z Zhang, G Kong, Journal of extracellular vesicles 8(1), 1643670). Bioinformatics analysis was carried out on the sequencing results, and the RNAs with differential expression in p...
Embodiment 2
[0045] Example 2 miRNA, mRNA and lncRNA detection system based on fluorescent quantitative PCR platform
[0046] 1. miRNA detection system
[0047] 1) miRNA reverse transcription reaction system
[0048] miRNA reverse transcription reagents, enzymes and oligdT were purchased from TAKARA, standard products were synthesized from Shanghai Yingwei Jieji, and specific reverse transcription primers were synthesized by Suzhou Synbio. A 20 μL reverse transcription system was used, as shown in Table 2.
[0049] Table 2 Reverse transcription system of miRNA
[0050]
[0051]
[0052] 2) qPCR reaction system:
[0053] The PCR reaction mixture was purchased from TAKARA, the upstream primers and probes, namely the universal downstream primers, were synthesized by Suzhou Synbio, and the fluorescent quantitative PCR instrument was ABI 7500. The PCR reaction system is shown in Table 3:
[0054] Table 3 qPCR reaction system of miRNA
[0055]
[0056] 2. mRNA and lncRNA detection...
Embodiment 3
[0078] Example 3 Effect Evaluation of Colorectal Adenoma Early Diagnosis Detection Kit
[0079] 1) Sample collection
[0080] 10 mL of blood was collected from hospital-diagnosed early (stage I and stage II) colorectal adenoma patients (24 cases), colorectal benign patients and healthy control samples (53 cases in total), and the plasma was separated.
[0081] 2) Exosomal RNA extraction
[0082] Plasma exosomes were separated using Qiagen’s commercial ExoEasy kit or Enze Kangtai’s Exosupur, and the isolated exosomes were extracted from exosomes with Qiagen miReasy mini kit, and the RNA concentration and mass, and record the RNA concentration.
[0083] 3) RNA two-step detection system
[0084] Using the mRNA and lncRNA detection system based on the fluorescent quantitative PCR platform in Example 2, detect the plasma exosomal RNA extracted in step 2), detect the Ct value of the target RNA, and calculate the relative expression level according to the Ct value and the relative...