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Identification of reference gene for detecting lung cancer miRNA

A lung cancer and internal reference technology, applied in the field of internal reference miRNA gene identification, to achieve the effect of improving sensitivity and specificity, and easy detection

Active Publication Date: 2018-05-25
深圳科诺医学检验实验室
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

To date, no studies have systematically identified and evaluated the best reference genes for miRNA normalization in lung cancer research. Therefore, there is an urgent need in the field to develop reference genes that can serve as microRNA normalization in lung cancer research and to establish methods for detecting miRNAs, especially circulating miRNAs. effective standardization

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  • Identification of reference gene for detecting lung cancer miRNA
  • Identification of reference gene for detecting lung cancer miRNA
  • Identification of reference gene for detecting lung cancer miRNA

Examples

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Effect test

example 1

[0024] Example 1 Analysis of miRNA expression in peripheral blood of patients with lung cancer

[0025] 1. Materials and methods

[0026] 1. Materials

[0027] Peripheral blood of lung cancer patients was obtained from hospitalized cases of lung cancer. Peripheral blood was collected from 30 lung cancer patients and 20 normal people.

[0028] 2. Method

[0029] 2.1. Extraction of total RNA from peripheral blood

[0030] According to the instructions of the Triazol kit, total RNA was extracted from the peripheral blood cells of lung cancer patients and normal people, the integrity of the RNA was verified by gel electrophoresis, and the concentration and purity of the RNA were determined with a nucleic acid protein analyzer. Proceed as follows:

[0031] (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 temperature for 5 minutes. Add 200 μL of chloroform, shake vigorously for 1...

Embodiment 2

[0041] The qRT-PCR verification of miRNA in embodiment 2 serum / plasma

[0042]According to the results of chip hybridization, miRNA molecules meeting the following criteria were selected for further screening by qRT-PCR technology: a) the expression levels in lung cancer and the control group were in the top 100; b) they were stably expressed in both groups, and There was no significant difference between them (p≥0.05). According to the above criteria, a total of 9 qualified miRNA molecules were selected (including hsa-miR-140-5p, hsa-miR-26a, hsa-miR-99b, hsa-miR-106b, hsa-miR-151-5p, hsa-miR-23a, hsa-miR-342-3p, hsa-miR-345-5p, hsa-miR-16-5p), the sequences of nine miRNA molecules are shown in Table 1. In addition, since U6 is often used as an internal reference molecule for tissue miRNA detection, U6 was also included in the screening as a candidate molecule in order to verify whether it can be used as an internal reference in serum. By using qRT-PCR technology to verify ...

Embodiment 3

[0059] Example 3 Further verification of expression stability

[0060] According to the results of qRT-PCR screening, has-miR-345-5p or the combination of has-miR-345-5p and has-miR-99b are candidate internal reference genes with stable expression. The qRT-PCR method was used to verify its stability in a new set of tested samples (including 12 liver cancer and 12 control samples), and the results showed that it was stably expressed in both liver cancer and normal samples, and had a good stability.

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Abstract

The invention discloses a reference gene miRNA (micro-ribonucleic acid) with stable expression effect in a patient with lung cancer. The reference gene comprises a combination of hsa-miR-345-5p or hsa-miR-345-5p and hsa-miR-99b. The reference gene has the advantages that the reference gene can be used for the standardizing of a target gene in miRNA detection of the patient with lung cancer, the study results of different laboratories can be compared, and the normalization processing of the experience results is promoted.

Description

technical field [0001] The patent of the present invention relates to the identification of an internal reference miRNA gene for lung cancer miRNA detection. 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), by 2025, the number of lung cancer deaths in my country will reach 1 million each year. In recent years, although great progress has been made in the diagnosis and clinical treatment of lung cancer, the overall survival time of patients is still very short, and the 5-year survival rate does not exceed 15%. The current treatment methods have little effect on improving the overall survival rate of lung cancer. The 5-year survival rate of patients with stage II-IV lung cancer is about 40%-5%, while the 5-year survival rate of patients with stage I lung cancer can ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886
CPCC12Q1/6886C12Q2600/158C12Q2600/166C12Q2600/178
Inventor 朱伟
Owner 深圳科诺医学检验实验室
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