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Method, primers, probes and kit for detecting relative expression of RBM5 gene

A technology of relative expression and RBM5-R, which is applied in the fields of life science and biology, can solve the problems of high cost and poor specificity, and achieve the effect of simple operation, easy access and good therapeutic effect

Inactive Publication Date: 2020-05-08
WUHAN ADICON CLINICAL LAB
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, because SYBRGreenI is an unsaturated dye, its specificity is not as good as that of the double-probe hybridization method and the Taqman method, and its specificity must be judged by observing the melting curve; and the cost of the double-probe hybridization method is relatively expensive.

Method used

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  • Method, primers, probes and kit for detecting relative expression of RBM5 gene
  • Method, primers, probes and kit for detecting relative expression of RBM5 gene
  • Method, primers, probes and kit for detecting relative expression of RBM5 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] The nucleic acid detection kit used to detect the relative expression of the RBM5 gene in samples can be used to evaluate the treatment effect of lung cancer, monitor the progress of lung cancer, and prompt recurrence as soon as possible. The kit includes: sample RNA extraction reagent; RNA reverse transcription reagent; detection system PCR reaction solution; positive control substance, negative control substance and blank control substance.

[0043] Among them, the sample RNA extraction reagent can extract RNA in tissue or blood. Sample RNA extraction reagents and RNA reverse transcription reagents can be prepared by yourself, or purchased from commercial reagents such as related kits from TOYOBO. For example, sample RNA extraction reagents can be purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. Kit RNAsimple T otal RNA Kit and RNA reverse transcription reagents can be purchased from Rever Tra Ace qPCR RT Kit Kit (TOYOBO Company).

[0044] Detection ...

Embodiment 2

[0053] 1. Preparation of blood sample RNA. Use the RNAsimple TotalRNA Kit of Tiangen Biochemical Technology (Beijing) Co., Ltd. to prepare RNA from blood samples. The preparation steps are as follows:

[0054] (1) Take 250 μl of fresh blood directly and add 3 times the volume of RZ;

[0055] (2) Place the homogenized sample at 15-30°C for 5 minutes to completely separate the nucleic acid-protein complex;

[0056] (3) Centrifuge at 12,000 rpm at 4°C for 5 minutes, take the supernatant, and transfer it to a new RNase-free centrifuge tube. Note: If the sample contains a lot of protein, fat, polysaccharide or muscle, plant nodules, etc., this step can be added to remove it by centrifugation. The precipitate obtained by centrifugation includes cell outer membrane, polysaccharide and high molecular weight DNA, and RNA exists in the supernatant solution.

[0057] (4) Add 200 μl of chloroform, cover the tube cap, shake vigorously for 15 sec, and place at room temperature for 3 min....

Embodiment 3

[0074] Example 3 Positive plasmid detection and sensitivity detection

[0075] When preparing a positive plasmid, first directly synthesize RBM5 cDNA and Actin cDNA, and then respectively insert the synthesized RBM5 cDNA into a previously selected plasmid (here, the PUC57-T plasmid is used as an example), so that the PUC57-T plasmid can be prepared. T / RBM5 positive plasmid and PUC57-T / Actin positive plasmid. The prepared PUC57-T / RBM5 positive plasmid was serially diluted to obtain a copy number of 10 8 、10 7 、10 6 、10 5 、10 4 、10 3 、10 2 , 10copies / μl of the positive plasmid, and with these different concentrations of the PUC57-T / RBM5 positive plasmid as a template, detect according to Example 2, the results are as follows figure 1 shown. In addition, the prepared PUC57-T / Actin positive plasmid was serially diluted to obtain a copy number of 10 8 、10 7 、10 6 、10 5 、10 4 、10 3 、10 2 , 10copies / μl positive plasmid.

[0076] exist figure 1 The PUC57-T / RBM5 positi...

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Abstract

The invention discloses a method, primers, probes and kit for detecting relative expression of an RBM5 gene, and all relates to the primers RBM5-F and RBM5-R and the probe RBM5-Probe for detecting theexpression level of the RBM5 gene, and the primers Actin-F, Actin-R and probe Actin-Probe for detecting reference gene Actin. The primers and probes provided by the invention have good specificity, high sensitivity and easy operation after tests; and the method, primers, probes and kit provided by the invention can quickly detect the amount of the RBM5 gene and the expression in a sample, and canassist in formulating individualized treatment plans and disease prognosis judgment for lung cancer patients.

Description

technical field [0001] The invention belongs to the field of life science and biotechnology, and in particular relates to a primer, a probe, a method and a kit for detecting the relative expression level of RBM5 gene. Fluorescent PCR technology is used to detect the expression level of RBM5 gene in tumor patients. Background technique [0002] Lung cancer is the most common malignant tumor worldwide, and 80% to 85% of the cases are small cell lung cancer (NSCLC). According to statistics, in Europe, there are more than 200,000 new cases every year, and the death toll accounts for 20% of all cancer-related deaths. In the United States, approximately 150,000 patients die of lung cancer each year. In mainland China, both the incidence and mortality of lung cancer have shown a significant upward trend. Although the diagnostic methods, surgical operations, chemotherapy drugs and radiotherapy methods are constantly updated and developed, the 5-year survival rate of lung cancer is...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6886C12Q2600/158C12Q2563/107C12Q2545/114C12Q2561/101
Inventor 李允章刘赵玲王淑一
Owner WUHAN ADICON CLINICAL LAB
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