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Detection primer group for identifying pluripotent stem cell endogenous and exogenetic pluripotent gene expression, kit and detection method thereof

A technology for pluripotent stem cells and gene expression, applied in the field of detecting primer sets for identifying endogenous and exogenous pluripotent gene expression in pluripotent stem cells, can solve the problem of identifying endogenous and exogenous pluripotency of pluripotent stem cells Gene expression kits and other issues

Inactive Publication Date: 2018-05-04
GUANGDONG XTEM BIOTECH CO LTD
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  • Abstract
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  • Claims
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Problems solved by technology

Although Real Time PCR technology has been widely used in gene quantitative analysis, there are no kits that use Real Time PCR technology to identify endogenous and exogenous pluripotent gene expression in pluripotent stem cells

Method used

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  • Detection primer group for identifying pluripotent stem cell endogenous and exogenetic pluripotent gene expression, kit and detection method thereof
  • Detection primer group for identifying pluripotent stem cell endogenous and exogenetic pluripotent gene expression, kit and detection method thereof
  • Detection primer group for identifying pluripotent stem cell endogenous and exogenetic pluripotent gene expression, kit and detection method thereof

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Embodiment Construction

[0041] Hereinafter, the present invention will be further described with reference to the drawings and specific embodiments to better understand the present invention.

[0042] 1. Experimental materials:

[0043] Human skin fibroblasts (HFF), hiPS cells (HFF-derived-iPSCs) induced by retroviral method.

[0044] 2. Primer screening

[0045] 1. Screening of primers for detection of endogenous pluripotent genes by fluorescence quantitative PCR for Oct4, Sox2 and Nanog

[0046] Design multiple pairs of primers with amplified fragments of about 150 bp for the endogenous genes Oct4, Sox2 and Nanog of pluripotent stem cells. According to the conventional Trizol method, the total RNA of hiPS cells was extracted and reverse transcribed into cDNA, and each pair of designed primers was used for amplification. Increase each pair of primers with 2 to 3 repeats.

[0047] The amplification system is as follows:

[0048] 2×SYBR Premix Ex Taq

10μL

Upstream and downstream primers

0.2μM each

ROX ...

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Abstract

The invention relates to a primer composite for identifying human induced pluripotent stem cell pluripotent genes, a kit and a detection method. The primer group comprises a primer group A, a primer group B and a confidential reference primer, wherein the primer group A comprises one or several kinds of primer pairs for amplifying human inducted pluripotent stem cell endogenous genes Oct4, Sox2 and Nanog; the primer group B comprises one or several kinds of primer pairs for amplifying Oct4, Sox2 and Nanog general genes; the confidential reference primer is a primer for amplifying a GAPDH gene.Concretely, RNA (ribonucleic acid) is extracted from the pluripotent stem cells; a real-time PCR(polymerase chain reaction) technology is used by the detection method and the kit for performing quantitative determination on the pluripotent gene so as to identify the endogenous and exogenetic pluripotent gene expression differences. By using the identification method provided by the invention, theoperation is simple; the cost is low; the accuracy and the sensitivity are high.

Description

Technical field [0001] The present invention relates to the field of biotechnology, in particular to a detection primer set, kit and detection method for identifying the expression of endogenous and exogenous pluripotency genes in pluripotent stem cells. Background technique [0002] Pluripotent Stem Cells (PSCs) are a class of cells that have self-renewal and differentiate into a variety of cell tissues, mainly including embryonic stem cells (Embryonic Stem Cells, ESCs) and induced pluripotent stem cells (iPSCs) . ESCs are obtained by isolating the inner cell mass of blastocysts or primordial germ cells. iPSCs are self-renewing and pluripotent cells obtained by expressing specific transcription factors in differentiated somatic cells to induce their reprogramming. The establishment of iPSCs technology avoids ethical barriers. It not only provides unique tools for the study of differentiation potential mechanisms and normal development in basic research, but also has great poten...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6851C12N15/11
CPCC12Q1/6851C12Q2531/113C12Q2545/101
Inventor 彭特于云飞陈勇蔡亚雄刘樱乔志平
Owner GUANGDONG XTEM BIOTECH CO LTD
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