Marker LINC01977 and application thereof

A biomarker and DNA sequence technology, applied in the biological field, can solve problems such as the inability to guarantee the concentration and purity of lncRNA

Active Publication Date: 2021-05-18
JIANGSU CANCER HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Existing RNA extraction kits cannot guarantee the concentration and purity

Method used

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  • Marker LINC01977 and application thereof
  • Marker LINC01977 and application thereof
  • Marker LINC01977 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Extraction of RNA from lung adenocarcinoma tissue, the specific steps are as follows:

[0083] (1) Preparation of lung adenocarcinoma tissue homogenate: Use a pipette to absorb 1 mL of sterile normal saline, wash the residual blood on the surface of lung adenocarcinoma tissue, cut it into 5 mm tissue pieces with ophthalmic scissors, and put them into pre-filled 1 mL Trizol Reagent Tissue Homogenizer Tube. Place the tissue homogenization tube into the slot of the homogenizer, 100rpm, 30s, take it out and put it on ice to cool down, repeat the above operation until no tissue blocks are visible to the naked eye. Remove the tissue homogenate tube and put it on ice for later use.

[0084] (2) Chloroform extraction: Transfer the liquid in the homogenization tube to a 1.5 mL centrifuge tube, add 500 uL of chloroform pre-cooled at 4°C, invert up and down evenly, and place at room temperature for 5 min. Centrifuge at 12000rpm for 15min at 4°C, carefully absorb the upper colorl...

Embodiment 2

[0090] Detection and identification of LINC01977 in lung adenocarcinoma tissue and its clinical diagnostic value

[0091] (1) Preparation of cDNA

[0092] Table 4 The reaction system for the preparation of cDNA

[0093]

[0094] Note: The amount of RNA template in this system should not exceed 2.5 μg, otherwise it will affect the result of the reaction.

[0095] Reverse transcription reaction conditions are as follows: 37°C, 15min; 85°C, 5sec; 4°C.

[0096] (2) Real-time fluorescent quantitative qPCR

[0097] Apply QuantStudio TM The 6Flex system performs qRT-PCR experiments on the cDNA prepared in step 1. The reaction system is as follows:

[0098] The qRT-PCR reaction systems containing LINC01977 and β-actin upstream and downstream primers were respectively configured as shown in Table 5.

[0099] Table 5 qRT-PCR reaction system

[0100]

[0101] The qRT-PCR reaction conditions are shown in Table 6.

[0102] Table 6 qRT-PCR reaction conditions

[0103]

[0...

Embodiment 3

[0113] In situ hybridization detection and pathological section analysis for LINC01977

[0114] The present invention utilizes early lung adenocarcinoma tissue chips with lymph node metastasis, including 98 cases of tumor tissues and 98 cases of paired paracancerous normal tissues (normal tissues around / beside the tumor), and uses lncRNA tissue in situ hybridization to detect LINC01977 in the tissues The expression level.

[0115] Method: Tissue in situ hybridization probes were designed according to the specific sequence of LINC01977, and the hybridization kits from ACD Company (Cat. No.: 322350, 323180) were used for experiments. The steps are as follows:

[0116] (1) slice preprocessing:

[0117] Melt wax at 60°C for 30 minutes; soak in xylene twice, 5 minutes each; soak in absolute ethanol twice, 1 minute each; incubate with hydrogen peroxide solution at room temperature for 10 minutes; wash with distilled water twice, 5 minutes each;

[0118] (2) Antigen retrieval:

[...

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Abstract

The invention belongs to the technical field of biology and the technical field of molecular diagnosis, and discloses a marker LINC01977 and application thereof. A kit for detecting lncRNA of lung adenocarcinoma tissue provided by the invention can conveniently and accurately detect long-chain non-coding RNA: LINC01977 from the tissue, is beneficial to analysis and application of LINC01977 extracted from lung adenocarcinoma tissue in laboratories and clinics; an LINC01977 detection method and kit provided can quickly detect LINC01977; a diagnostic kit for early lung adenocarcinoma prone to lymph node metastasis can quickly detect LINC01977; and experiments prove that the LINC01977 in lung adenocarcinoma tissues can be used as a novel marker for judging whether early lung adenocarcinoma has a lymph node metastasis tendency or not and has a prognosis judgment value. The invention provides help for clinical treatment such as recurrence, metastasis, prognosis evaluation and the like of lung adenocarcinoma.

Description

technical field [0001] The invention belongs to the field of biological technology and molecular diagnosis technology, and relates to a lung adenocarcinoma tissue marker LINC01977, a detection method and an application. Background technique [0002] Lung cancer (lung cancer) is one of the malignant tumors with the highest morbidity and mortality in the world, and has become a huge medical burden worldwide. my country is a country with a high incidence of lung cancer, and its incidence ranks first among all malignant tumors in my country. The most common pathological type is lung adenocarcinoma. At present, the diagnosis of lung adenocarcinoma depends on tissue biopsy. Patients with early lung adenocarcinoma are diagnosed by surgery and pathology, and their prognosis is good. Lymph node metastasis in patients with early lung adenocarcinoma is often accompanied by a worse prognosis. The main treatment for lung adenocarcinoma prone to recurrence and metastasis is surgical rese...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12N15/113
CPCC12Q1/6886C12Q2600/178C12Q2600/158C12Q2600/118
Inventor 董高超蒋峰张特宋绪鸣张泽宇李如涛夏文杰毛启星
Owner JIANGSU CANCER HOSPITAL
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