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MiRNA combination used for detecting esophageal squamous cell carcinoma and application thereof

A cell carcinoma, esophageal squamous cell technology, applied in recombinant DNA technology, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problems of tissue and cell detection defects, lack of human experience, improper material selection, etc., to achieve simple and easy effects. , easy to use, improve the accuracy of the effect

Inactive Publication Date: 2010-10-06
NANJING GENERAL HOSPITAL NANJING MILLITARY COMMAND P L A +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The current clinical diagnostic methods include imaging, endoscopic examination, tissue biopsy, esophageal mucosal exfoliative cytology, etc., but these methods have certain defects in early diagnosis: imaging is useful for the diagnosis of early esophageal squamous cell carcinoma. Certain limitations, while tissue cell detection has its inherent defects, improper sampling or lack of human experience will lead to misdiagnosis
[0005] At present, there are no specific miRNA combinations and detection kits related to esophageal squamous cell carcinoma using non-invasive and convenient blood sample detection

Method used

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  • MiRNA combination used for detecting esophageal squamous cell carcinoma and application thereof
  • MiRNA combination used for detecting esophageal squamous cell carcinoma and application thereof
  • MiRNA combination used for detecting esophageal squamous cell carcinoma and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0056] Example 1: Preliminary screening of specific miRNA expression profiles in esophageal squamous cell carcinoma

[0057] (1) 86 cases, 55 cases and 40 cases of serum samples of non-metastatic, metastatic esophageal squamous cell carcinoma and normal subjects were collected respectively, and the samples of the three groups were mixed respectively;

[0058] (2) Extract the RNA in the mixed serum of the three groups respectively. The specific plan is: use Trizol reagent (Invitrogen Company) to extract the total RNA, and use the phenol-chloroform three-step method to further remove impurities such as protein and purify the RNA from the supernatant;

[0059] (3) Solexa sequencing analysis was performed on the total RNA of the mixed serum of the three groups.

[0060] The specific plan is: Solexa sequencing is based on single-molecule cloning array technology, which can distinguish the difference of a single base at the level of the entire genome, thereby distinguishing the diff...

Embodiment 2

[0067] Embodiment 2: Real-time fluorescent quantitative PCR experiment (TaqMan probe method) of miRNA in serum

[0068] Quantitative PCR was used to verify batches of samples one by one, and miRNAs with significant expression differences were screened from the miRNAs initially screened by the Solexa method.

[0069] Firstly, the extracted serum total RNA was reverse transcribed into cDNA. For each miRNA, design a gene-specific reverse primer containing the same stem-loop structure, use the miRNA-specific reverse primer to perform reverse transcription, and obtain a cDNA (product of ABI Company) that contains a common stem-loop structure but belongs to a specific miRNA.

[0070] The instrument used is ABI Prism 7300 fluorescent quantitative PCR instrument.

[0071] In this experiment, different concentrations of artificially synthesized mature miR-16 were used as a standard curve. The artificially synthesized miR-16 stock solution was reverse-transcribed into cDNA, and dilute...

Embodiment 3

[0075] Embodiment 3: Quantitative PCR method re-screens the serum miRNA screened out

[0076] A group of subject samples were detected one by one by TaqMan quantitative PCR method, and miRNAs with significant expression differences between the patient group and the control group were screened from the 25 miRNAs initially screened by the Solexa method (see Example 2 for specific steps). There were 36 patients with esophageal squamous cell carcinoma in this group, and 33 cancer-free patients matched in age and gender were used as controls (see Table 2 for detailed clinical information of patients). It was found that among the 25 miRNAs initially screened out, the expression levels of 7 miRNAs in the patient group were significantly higher than those in the control group (the expression method of content is mean ± standard deviation; the multiple of change is 1.65-2.97 times, p<0.0001). The seven miRNAs are miR-10a, miR-22, miR-100, miR-148b, miR-223, miR-133a and miR-127-3p (see...

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Abstract

The invention belongs to the technical field of biotechnology, and discloses a miRNA combination used for detecting esophageal squamous cell carcinoma and application thereof. The miRNA combination comprises miR-22, miR-127-3p, miR-148b and miR-223, and the miRNA combination can further comprise one or more of miR-10a, miR-100 and miR-133a. A kit comprises a probe combination of the miRNAs. The miRNA combination and the probe combination thereof provided by the invention can be used for detecting the esophageal squamous cell carcinoma, and can be used as a diagnosis marker for improving the accuracy of detection, improving the insuperable low specificity and low sensitivity, caused by individual difference, of a single marker, and improving a clinical detectable rate remarkably by combining other detection indicators and clinical symptoms of a patient, so the miRNA combination and the probe combination thereof become an effective means for early diagnosis of the esophageal squamous cell carcinoma. Due to the adoption of the miRNA combination, a noninvasive serological test is adopted so as to bring convenience to clinical promotion.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to the application of microRNA (miRNA for short), in particular to a miRNA combination for detecting esophageal squamous cell carcinoma, and a TaqMan probe combination corresponding to the miRNA combination Reagent test kit. Background technique [0002] Esophageal squamous cell carcinoma is a malignant tumor that originates in the esophagus, accounting for more than 90% of esophageal cancers. Progressive dysphagia is its typical clinical manifestation. Esophageal squamous cell carcinoma has two high and one low characteristics of high morbidity, high mortality and low detection rate. At present, the incidence of esophageal squamous cell carcinoma ranks eighth among all kinds of tumors in the world, and the mortality rate ranks sixth. There are 410,000 new cases of esophageal squamous cell carcinoma every year in the world, and about 310,000 deaths. my country is an area with a high inc...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 张春妮王成吕志远王琛陈熹张辰宇
Owner NANJING GENERAL HOSPITAL NANJING MILLITARY COMMAND P L A
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