MiRNA detection kit for lung cancer prognostic prediction

A kit and prognostic technology, applied in the field of detection of a group of peripheral blood miRNAs, can solve the problems of high equipment requirements, low sensitivity and poor specificity, etc.

Inactive Publication Date: 2015-11-25
陈建华
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  • Abstract
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  • Claims
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Problems solved by technology

[0005] At present, tumor markers and other non-invasive screening methods have the problems of low sensitivity, poor specificity, and high requ...

Method used

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  • MiRNA detection kit for lung cancer prognostic prediction
  • MiRNA detection kit for lung cancer prognostic prediction
  • MiRNA detection kit for lung cancer prognostic prediction

Examples

Experimental program
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Effect test

example 1

[0020] Example 1 Establishment of miRNA biomarker combination in peripheral blood of lung cancer patients

[0021] 1. Materials and methods

[0022] 1. Materials

[0023] The peripheral blood of patients with lung cancer was obtained from hospitalized cases of lung cancer in Wuhan University People's Hospital from 2012 to 2014. Peripheral blood was collected from 30 lung cancer patients and 20 normal people.

[0024] 2. Method

[0025] 2.1. Extraction of total RNA from peripheral blood

[0026] According to the instructions of the Triazol kit, the total RNA of the peripheral blood cells of lung cancer patients was extracted, the integrity of the RNA was verified by gel electrophoresis, and the concentration and purity of the RNA were determined by a nucleic acid protein analyzer. The main steps are as follows:

[0027] (1) Collect peripheral blood from the patient, collect cells by centrifugation, add 1 mL of Trizol reagent to each tube, mix well, and place at room tempera...

example 2

[0039] Example 2 Application of miRNA: hsa-miR-10b-5p, hsa-miR-141-3p to the method for detecting lung cancer patients

[0040] 1. Collect peripheral blood samples from patients with lung cancer.

[0041] 2. Extract total RNA according to the method of example 1.

[0042] 3. Reverse transcription and Realtime-PCR

[0043] Take 100ng of the extracted RNA and perform reverse transcription using the ReverseTranscription reverse transcription kit from Promega Company. 20μL reaction system: 100ng extracted RNA, 4μL 5×RTbuffer, 1μL AMVreversetranscriptasemix, make up to 20μL with Rnasefreewater. Reverse transcription was carried out on a PCR instrument under the following conditions: 37°C for 1 hour and 95°C for 5 minutes.

[0044] Take 1.5 μL of the reverse transcription product as the template of the real-time quantitative PCR reaction, and use Qigan’s SYBRGreenPCR kit to perform real-time quantitative PCR, and the reaction uses U6snRNA as the internal reference. The reaction sy...

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Abstract

The invention relates to a kit for detecting peripheral blood miRNA variation of lung cancer, and particularly relates to a kit and a use method thereof. The kit comprises the following components: a specific primer, a specific probe, a U6snRNA primer and a fluorescence PCR reaction liquid. The detection method comprises the following steps: collecting a peripheral blood sample of a patient and extracting total RNA in the blood; carrying out real-time quantitative PCR by the kit, so that expression quantities of hsa-miR-10b-5p and hsa-miR-141-3p are rapidly detected in large scales; and comparing with internal reference U6snRNA, thus realizing prognostic prediction on a lung cancer patient. The kit is easy and rapid to operate, good in repeatability, strong in specificity, and high in sensitivity.

Description

technical field [0001] The invention belongs to the technical field of medical diagnosis, relates to the field of prognosis of lung cancer, mainly relates to the prediction of the risk of death after chemotherapy of lung cancer, and particularly relates to a detection method and application of a group of peripheral blood miRNAs for lung cancer diagnosis. Background technique [0002] Lung cancer is the leading cause of cancer death worldwide. In China, the incidence of lung cancer is increasing year by year, and its mortality rate ranks first among cancer deaths. According to the World Health Organization (WHO), as of 2025, my country's annual lung cancer deaths will reach 1 million (JemalA, BrayF, CenterMM, etal.Globalcancerstatistics.CACancerJClin, 2011, 61 (2): 69-90; HongQ, et al. Lungcancer China: challenges and interventions. Chest, 2013, 143(4): 1117-1126.). In recent years, although great progress has been made in the diagnosis and clinical treatment of lung cancer, ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 陈建华张礼斌
Owner 陈建华
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