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FISH probe composition and kit for detecting miRNA gene related to esophagus cancer and use method of kit

A composition and a technique for esophageal cancer are applied in the field of molecular biology to achieve the effects of easy collection, obvious specificity and improved detection efficiency

Inactive Publication Date: 2014-06-18
GUANGZHOU BIOSENSE BIOSCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In summary, there is no FISH technology in this field to detect the gene marker miRNA gene of esophageal cancer in saliva, so this technology can be developed and applied to the early screening of esophageal cancer

Method used

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  • FISH probe composition and kit for detecting miRNA gene related to esophagus cancer and use method of kit
  • FISH probe composition and kit for detecting miRNA gene related to esophagus cancer and use method of kit
  • FISH probe composition and kit for detecting miRNA gene related to esophagus cancer and use method of kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1: Design and synthesis of probes.

[0062] 15 miRNA genes related to esophageal cancer were analyzed through the online database miRBase (http: / / www.mirbase.org / ), and 18-25bp fluorescein-labeled probes were designed to detect the genes and their probe sequences as follows:

[0063] (1) Probe for hsa-miR-144 gene: 5'-CTTACAGTATATGATGATATCC-3 (SEQ ID NO.1);

[0064] (2) Probe for hsa-miR-1180 gene: 5'-TATTCCCGGCCGGGTGGGTCC-3 (SEQ ID NO.2);

[0065] (3) Probe for hsa-miR-196b gene: 5'-CCCAACAACAGGAAACTACCTA-3' (SEQ ID NO.3);

[0066] (4) Probe for hsa-miR-149 gene: 5'-GGAGTGAAGACACGGAGCCAGA-3' (SEQ ID NO.4);

[0067] (5) Probe for hsa-miR-1825 gene: 5'-GGAGAGGAGGGCACTGGA-3' (SEQ ID NO.5);

[0068] (6) Probe for hsa-miR-760 gene: 5'-TCCCCACAGACCCAGAGCCG-3' (SEQ ID NO.6);

[0069] (7) Probe for hsa-miR-135b gene: 5'-TCACATAGGAATGAAAAGCCATA-3' (SEQ ID NO.7);

[0070] (8) Probe for hsa-miR-139 gene: 5'-ACTCCAACAGGGCCGCGTCTCCA-3' (SEQ ID NO.8);

[0071] (9) Pro...

Embodiment 2

[0080] Embodiment 2: the preparation of reaction reagent.

[0081] The FISH probe composition used in the FISH kit is made into a solution, which is obtained by dissolving the FISH probe in 2x SSC buffer solution, the probe concentration is 5ng / μl, and the 2×SSC buffer solution is based on chloride Sodium chloride 8.8g, sodium citrate 4.4g, deionized water 400ml prepared.

[0082] In addition, the FISH kit also includes 300 μl of 5 mg / ml 4',6-diamidino-2-phenylindole (DAPI) counterstain solution.

Embodiment 3

[0083] Example 3: The experiment of detecting the positive expression of miRNA gene related to esophageal cancer in the saliva samples of patients with esophageal cancer by FISH.

[0084] Follow the steps below to test:

[0085] Including the following steps:

[0086] 1. Fix the saliva sample to be tested on the anti-shedding glass slide, and pretreat the saliva sample on the slide. The specific operation is as follows:

[0087] 1.1. Centrifuge the freshly collected saliva at 1500rpm for 8min.

[0088] 1.2. Remove the supernatant and add 5ml of 0.075M kcl hypotonic solution. Mix well, and then put in a 37°C water bath for 30min. (Effect: Use the osmotic pressure difference to rupture the cell membrane, and the cell nucleus swells when it absorbs water. It is conducive to the hybridization of the probe.)

[0089] 1.3. Add 2ml of fixative solution for pre-fixation (methanol: glacial acetic acid = 3:1), mix well and centrifuge at 1500rpm for 8min. (In the fixative, the smal...

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Abstract

The invention relates to a FISH probe composition for detecting a miRNA gene related to esophagus cancer. The probe composition contains a FISH probe for detecting the miRNA gene related to esophagus cancer. The invention further provides a FISH kit for detecting the miRNA gene related to esophagus cancer. The FISH kit comprises the FISH probe composition according to claims 1-5. In addition, the invention further discloses a use method of the FISH kit for detecting the miRNA gene related to esophagus cancer. According to the technical scheme, various miRNA genes related to esophagus cancer in saliva can be detected by directly adopting the fluorescence in situ hybridization technology, so that not only is a sample directly in-situ quantified, but also noninvasive diagnosis and screening of early esophageal cancer can be realized.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a FISH probe composition, kit and application method for detecting miRNA genes related to esophageal cancer. Background technique [0002] MicroRNAs (miRNAs) are a class of endogenous non-coding small molecule RNAs with regulatory functions discovered in recent years, with a size of about 20-25 nucleotides. miRNA genes are transcribed by RNA polymerase II in the nucleus to form primary transcripts (pri-RNAs). After a series of processing, Pri-RNA becomes a mature miRNA of about 19-25bp. Mature miRNAs can combine with proteins to form RNA-induced silencing complex (RISC) in cells. When RISC is not completely paired with the target mRNA gene, it can block the translation process of miRNA and reduce the expression; when RISC is completely paired with the target mRNA, it can lead to the degradation of mRNA. miRNA controls the expression of target genes by blocking or degrading the...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6841C12Q2525/207C12Q2543/10C12Q2563/107
Inventor 吴伟东王晓香周剑董先辉刘雪媚
Owner GUANGZHOU BIOSENSE BIOSCI
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