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Kit for detecting relative expression of PDGFR alpha gene by real-time fluorescent quantitative PCR

A real-time fluorescence quantification and kit technology, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problems such as the inability to estimate the amount of initial template, the variety of reagents, the cumbersome test process, etc., to avoid hematology. Recurrence, results are easy to interpret, the effect of evaluating the effect of treatment

Inactive Publication Date: 2019-06-04
南昌艾迪康医学检验实验室有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the results of the FISH method are relatively intuitive, the test process is cumbersome, involving a wide variety of reagents, time-consuming and laborious, and the results need to be interpreted by experienced professionals, and the result interpretation is relatively subjective.
However, the simple RT-PCR method is quantitative at the end point, and cannot accurately estimate the amount of starting template.

Method used

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  • Kit for detecting relative expression of PDGFR alpha gene by real-time fluorescent quantitative PCR
  • Kit for detecting relative expression of PDGFR alpha gene by real-time fluorescent quantitative PCR
  • Kit for detecting relative expression of PDGFR alpha gene by real-time fluorescent quantitative PCR

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] The kit is used to assist clinical early prevention, early diagnosis and auxiliary indicators of prognosis of human tumors; it can also perform more accurate screening for high-risk groups. Kit includes:

[0026] 1. Tissue RNA extraction reagent;

[0027] 2. Blood RNA extraction reagent;

[0028] 3. RNA reverse transcription reagent

[0029] 4. Detection system PCR reaction solution;

[0030] 5. Positive control substance and negative control substance.

[0031] Among them, tissue and blood RNA extraction reagents and RNA reverse transcription reagents can be purchased from commercial reagents such as related kits from TOYOBO.

[0032] Detection system PCR reaction solution: ReverTra Ace qPCR RT Kit (TOYOBO); TH UNDERBIRDProbe qPCR Mix (2×), PDGFRα upstream and downstream primers, PDGFRα probe; β-actin upstream and downstream primers, β-actin-probe probe ; Wherein the primer and probe sequences are:

[0033] PDGFRα-F: CGATTGTGGTCACCTGTGC

[0034]PDGFRα-R: TGGATGG...

Embodiment 2

[0041] The operation flow of this method:

[0042] 1. Preparation of RNA (paraffin). The extraction steps are as follows:

[0043] 1.1 Cut off the tissue or paraffin sample in a 1.5ml centrifuge tube (scrape)

[0044] 1.2 Add 1ml tissue clear solution, shake and mix well, then centrifuge at 13000rpm for 1min

[0045] 1.3 Remove the supernatant and add 500ml tissue clear solution, shake and mix well, then centrifuge at 13000rpm for 1min

[0046] 1.4 Remove the supernatant, add 1ml of absolute ethanol, shake and mix, then centrifuge at 13000rpm for 1min

[0047] 1.5 After removing the supernatant, put it in a 37-degree metal bath for 10 minutes (open the cover) until the liquid is dry

[0048] 1.6 Add 150ulbuffer PKD (from RNeasy FFPE KIT) and shake and mix at 1000rpm for 1min

[0049] 1.7 Add 10ulPK (from RNeasy FFPE KIT) and mix upside down

[0050] 1.8 15 minutes in a metal bath at 56°C, then 15 minutes in a metal bath at 80°C

[0051] 1.9 After 3 minutes on ice, 13200...

Embodiment 3

[0061] 1. Refer to the instruction manual of the Rever Tra Ace qPCR RT Kit kit from TOYOBO to reverse RNA to cDNA.

[0062] 2. Reagent configuration: according to the number of people to be tested, X ul of each PCR reaction solution of the detection system is configured, and each person is divided into 23ul:

[0063] X=23ul reaction solution×(8 internal references (standard curve)+8 target genes (standard curve)+n samples+1 positive control+1 negative control+1 blank control);

[0064] 3. Adding samples: add 2ul cDNA to the PCR reaction solution of the detection system; directly add 2ul positive control substance and negative control substance to the positive control and negative control; add 2ul normal saline or no substance to the blank control.

[0065] 4. Detection: detection is carried out on a real-time fluorescent PCR instrument, available instruments include ABI7300, 7500 (Applied Biosystems, USA) and the like. Reaction conditions: pre-denaturation at 95°C for 1min; 4...

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Abstract

The invention discloses a kit for detecting relative expression of PDGFR alpha gene by real-time fluorescent quantitative PCR. The kit comprises an RNA extraction reagent, a reverse transcription reagent, a detection system PCR reaction solution, a positive control substance and a negative control substance, wherein the detection system PCR reaction solution comprises sense and anti-sense primersPDGFR alpha-F and PDGFR alpha-R and probe PDGFR alpha-Probe for detecting target genes, and primers beta-actin-F and beta-actin-R and probe beta-actin-Probe for detecting reference gene beta-actin. The kit for detecting relative expression of PDGFR alpha gene by real-time fluorescent quantitative PCR combines real-time fluorescent PCR technology with a Taqman probe to separately construct quantitative standard curves of the reference gene beta-actin and the target gene PDGFR alpha by using a double standard curve method, and can detect the expression level of PDGFR alpha relative to the internal reference gene in a subject.

Description

technical field [0001] This patent relates to a detection kit for the relative expression of genes used in clinical testing. It uses probe Taqman real-time fluorescent quantitative PCR technology to detect the expression level of PDGFRα gene in tumor patients, and judge the curative effect and prognosis of drugs by the level of expression. . The kit can effectively save detection time and improve detection accuracy. Background technique [0002] The proposal of multi-target inhibition theory (multi-target inhibition of EGFR, VEGFR, FGFR, PDGFR, etc.) and the development of new drugs have made significant progress in the research of growth factor receptors related to tumor targeted therapy, among which platelet-derived growth factor PDGFR (Platelet -derived growth factor receptor (PDGFR) has become a new research hotspot after EGFR and VEGF / VEGFR. [0003] PDGFR is a single-chain transmembrane protein with a molecular weight of 180KD. The outside of the cell is divided into...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12Q1/6851
Inventor 王淑一吴鹏飞
Owner 南昌艾迪康医学检验实验室有限公司
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