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A primer composition for detecting the expression level of human-derived hsp70-1 gene and its application

A technology of gene expression level and primer composition, which is applied in the field of genes, can solve the problems of reduced expression level and unsuitable reference genes, etc., and achieve the effects of assisting research, avoiding interference of homologous genes, and simple design

Active Publication Date: 2020-03-24
SHANGHAI TONGJI HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Some normally expressed genes will be inhibited during the stress response of cells. Studies have shown that the commonly used actin housekeeping gene is affected by endoplasmic reticulum stress, and its expression level will be reduced. Therefore, it is not suitable to be used as a reference for endoplasmic reticulum stress. Gene

Method used

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  • A primer composition for detecting the expression level of human-derived hsp70-1 gene and its application
  • A primer composition for detecting the expression level of human-derived hsp70-1 gene and its application
  • A primer composition for detecting the expression level of human-derived hsp70-1 gene and its application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] This embodiment is a method for using the primer composition, which specifically includes the following steps:

[0037] Step 1: Tissue or Cell RNA Extraction

[0038] A commercial kit was used to extract cellular RNA, the concentration of RNA was measured by an ultraviolet spectrophotometer, and the quality of extraction was controlled. The measured value of OD260 / 280 was 2.0. In this example, the Tiangen total RNA extraction kit was used to extract cellular RNA, and the specific operation was performed according to the instructions.

[0039] Step 2: cDNA reverse transcription

[0040] About 500 ng of RNA was taken, and the cDNA was reverse-transcribed with the Takara commercial kit PrimeScriptTM RT Master Mix (Perfect Real Time), and the specific operation was performed according to the instructions.

[0041] Step 3: Real-time quantitative PCR reaction

[0042] In the experiment, two fluorescent quantitative PCR reaction systems were added to each template to detect H...

Embodiment 2

[0053] In this embodiment, the RNA interference sequence is screened according to the expression level of the HSP70-1 gene, and the specific steps are as follows:

[0054] Step 1: Cloning four different RNA interference sequences (S1-S4) of the HSP70-1 gene into lentiviral plasmid vectors, and transducing 293T cells by transient transduction method:

[0055] a. Culture 293T cells in high-glucose DMEM medium containing 10% FBS, and place the cells at 37°C, 5% CO 2 cultured in a cell culture incubator. 293T cells were digested 1 day before transduction, counted, and seeded into 35mm culture dishes, each culture dish was seeded with 6×10 5 cells;

[0056] b. The next day, use DMEM medium without FBS to prepare calcium transfer reagent and RNA interference plasmid respectively, add 16 μl calcium transfer reagent to each 84 μl medium, add 4 μg plasmid to each 100 μl medium, mix well and transfer the calcium transfer solution Gently mix with the plasmid solution, let stand at roo...

Embodiment 3

[0063] In this example, the expression level of human-derived HSP70-1 gene in lentivirus-infected cells is analyzed, and the specific steps are as follows:

[0064] Infect SH-SY5Y cells with lentivirus containing the HSP70-1 gene interference sequence: SH-SY5Y cells were placed in high-glucose DMEM medium containing 10% FBS at 37°C, 5% CO 2 Culture in an incubator, digest and count SH-SY5Y cells in logarithmic growth phase, inoculate into 96-well cell culture plate, 5000 cells / well, use DMEM medium without FBS to dilute RNAi lentivirus concentration the next day solution, add 10 μl lentivirus concentrate containing RNA interference sequence and 90 μl DMEM medium without FBS to each well, at 37°C, 5% CO 2 Cultivate in the incubator for 24 hours, replace with fresh high-sugar DMEM medium containing 10% FBS and continue to cultivate for 48 hours, collect cells to extract RNA, and use fluorescent quantitative PCR to detect the expression level of HSP70-1 for further verification a...

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Abstract

The invention discloses a primer composition for detecting expression level of human-derived HSP70-1 gene and application of the primer composition. The primer composition disclosed herein comprises primer group 1 and primer group 2; the primer group 1 is used for detecting HSP70-1 gene, and the primer group 2 is used for detecting RPL19 reference gene. The invention also relates to application of the primer composition in the preparation of reagents for detecting the expression level of human-derived HSP70-1 gene. Compared with the prior art, the primer composition according to the technical scheme and application thereof have the advantages that the expression level of HSP70-1 gene can be specifically detected, disturbance of homologous genes is avoided, detection sensitivity is very high, fluorescence probes are not required, the design is simple, the detection process is accurate and efficient, the detection time is short, and the primer composition has a promising application prospect.

Description

technical field [0001] The invention belongs to the field of gene technology, in particular to a primer composition for detecting the expression level of human HSP70-1 gene and its application. Background technique [0002] Heat shock proteins are a family of highly conserved protein molecules in the process of biological evolution. Members of this family can be divided into several categories such as HSP100, HSP90, HSP70, HSP60, and HSP28 according to the molecular weight. HSP70 is a heat shock protein with a molecular weight of about 70KD. It is the most conserved and important member of the heat shock protein family. It is called the main heat shock protein. They are encoded by the HSP70 gene family and are a non-specific cell protection protein. HSP70 can be divided into structural HSP70 and inducible HSP70. Structural HSP70 is produced by cells under normal conditions, and inducible HSP70 is produced by cells when they are stimulated, and is only slightly expressed or ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6883C12N15/11
CPCC12Q1/6851C12Q1/6886C12Q2600/118C12Q2600/158C12Q2531/113C12Q2545/101C12Q2563/107
Inventor 靳令经蒋明于丽华符星
Owner SHANGHAI TONGJI HOSPITAL
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