Real-time fluorescence quantitative PCR (polymerase chain reaction) special primer and method for detecting GDNF (glial cell line-derived neurotrophic factor) splice variants 2,4

A real-time fluorescence quantitative and fluorescent quantitative technology, applied in the fields of biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., to achieve the effect of simple operation, good repeatability and strong specificity

Inactive Publication Date: 2014-03-05
XUZHOU MEDICAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

According to the detection of the existing technology, there are no reports on the real-time fluorescent quantitative PCR primers and methods for the detection of GDNF splice variants 2 and 4 (that is, pre-(β)pro-GDNF) at home and abroad

Method used

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  • Real-time fluorescence quantitative PCR (polymerase chain reaction) special primer and method for detecting GDNF (glial cell line-derived neurotrophic factor) splice variants 2,4
  • Real-time fluorescence quantitative PCR (polymerase chain reaction) special primer and method for detecting GDNF (glial cell line-derived neurotrophic factor) splice variants 2,4
  • Real-time fluorescence quantitative PCR (polymerase chain reaction) special primer and method for detecting GDNF (glial cell line-derived neurotrophic factor) splice variants 2,4

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] The design of specific primers for the detection of pre-(β)pro-GDNF expression was based on the special sequence of pre-(β)pro-GDNF mRNA described in the research background, and the Primer Premier5.0 software was used to design primer sequences. Synthesized by Shanghai Sangon Biological Co., Ltd.

[0028] The primer sequence that the present invention obtains is:

[0029] Upstream primer: CCGCCGGACGGGACTTTA (SEQ ID NO.1),

[0030] Downstream primer: ATATTTGCGGCGGGCAGC (SEQ ID NO. 2).

Embodiment 2

[0031] Example 2 RNA extraction

[0032] Add TRIzol (Invitrogen Company) to the culture plate for cultivating U251 cells to lyse the cells, every 10 cm2 Add 1ml Trizol to the area, pipette, let stand at room temperature for 5min, add 0.2ml chloroform, mix upside down for 15s, let stand for 2min, centrifuge at 10,000g for 10min at 4°C, and transfer the upper aqueous phase to another clean EP tube Add 0.5ml of isopropanol, mix the liquid in the tube gently, let stand at room temperature for 10min, centrifuge at 10000g for 10min at 4°C, discard the supernatant; add 1ml of 85% ethanol to wash the precipitate. Centrifuge at 5000g for 30s at 4°C, discard the supernatant; air dry, add 30μl of DEPC H 2 O dissolved.

Embodiment 3

[0033] Example 3 mRNA reverse transcription: Reverse transcription was performed according to the instructions of the 1st strand cDNA synthesis kit (TAKARA D6210S).

[0034] 1st-Strand cDNA synthesis reaction

[0035] 1. Prepare the following reaction mixture in Microtube: Oligo dT Primer (50μM) 1μl, dNTP Mixture (10mM each) 1μl, Total RNA: less than 5μg, add RNase free dH2O to 10μl.

[0036] After incubation at 2.65°C for 5 minutes, it was cooled rapidly on ice.

[0037] 3. Prepare the following reverse transcription reaction solution in the above-mentioned Microtube tube, the total amount is 20 μl. 10 μl of the above-mentioned reaction solution after denaturation, 4 μl of 5×PrimeScript II Buffer, 0.5 μl (20U) of RNase Inhibitor (40U / μl), 1 μl (200U) of PrimeScript II RTase (200U / μl), and RNase free dH2O to 20 μl. Mix slowly.

[0038] 4. Perform the reverse transcription reaction under the following conditions: 30°C for 10 minutes, 42°C for 40 minutes, 95°C for 5 minutes, ...

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Abstract

The invention relates to a real-time fluorescence quantitative PCR (polymerase chain reaction) special primer and method for detecting GDNF (glial cell line-derived neurotrophic factor) splice variants 2,4. The sequences of the primer are shown in SEQIDNO.1 and SEQIDNO.2. The fluorescence quantitative PCR method is implemented by taking a brain tissue cDNA (complementary deoxyribonucleic acid) sample as a template, setting a template-free control, and groping and optimizing condition parameters of a quantitative reaction system by using the primer, so that a reliable and sensitive quantitative detection method for splice variants is developed. According to the invention, the expression levels of a GDNF splice variant 2 (GenecodeNO.: NM_001190469.1) and a splice variant 4 (GenecodeNO.: NM_199231.2) can be detected; the primer and method disclosed by the invention have the advantages of strong specificity and high repeatability.

Description

technical field [0001] The invention belongs to the field of biological detection, in particular to a real-time fluorescent quantitative PCR method for detecting GDNF splicing variants, in particular to a method for detecting GDNF splicing variant 2 (Gene code NO.: NM_001190469.1) and splicing variant 4 (Gene code NO.: NM_199231.2) expression level primers. Background technique [0002] Glial cell-line derived neurotrophic factor (GDNF) is a protein with a long history of research. It was originally isolated in the culture medium of rat glioma cell line B49 and is considered to maintain Neuronal development and regeneration, promoting the growth of neuronal axons (Lin et al.1993). From the perspective of protein structure, GDNF is a member of the transforming growth factor-β superfamily (transforming growth factor-β, TGF-β) (Airaksinen and Saarma, 2002). So far, researchers from various countries have studied it for nearly 20 years, and have also carried out various explor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/686C12Q2561/113C12Q2563/107
Inventor 李亨高殿帅张宝乐柴祥何涛
Owner XUZHOU MEDICAL COLLEGE
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