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Primer for quantitatively determining Twist-1 gene expression quantity in mesenchymal stem cells, standard product and detection method

A gene expression quantity and quality stem cell technology, applied in the biological field, achieves the effects of high accuracy, good primer specificity and low cost

Inactive Publication Date: 2018-10-23
CHENGDU ZHONGCHUANG QINGKE MEDICAL LAB CO LTD
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Problems solved by technology

[0004] With the upgrading of gene expression detection technology, there are higher requirements for the sensitivity and accuracy of the results, while conventional detection technology is difficult to meet the requirements of today's research

Method used

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  • Primer for quantitatively determining Twist-1 gene expression quantity in mesenchymal stem cells, standard product and detection method
  • Primer for quantitatively determining Twist-1 gene expression quantity in mesenchymal stem cells, standard product and detection method
  • Primer for quantitatively determining Twist-1 gene expression quantity in mesenchymal stem cells, standard product and detection method

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Embodiment 1

[0033] 1. Design primers

[0034] According to the sequence information of the Twist-1 gene in the NCBI database (its sequence is shown in SEQ ID NO: 3), the present invention has designed primers with excellent specificity for the expression level of the Twist-1 gene, and the primer sequences are as follows:

[0035] Upstream primer 5'-GTCCGCGTCCCACTAGC-3' (SEQ ID NO: 1);

[0036] Downstream primer 5'-TCCATTTTCTCCTTCTCTGGAA-3' (SEQ ID NO: 2);

[0037] The amplified length is 136bp.

[0038] 2. Total RNA extraction and reverse transcription

[0039] The mesenchymal stem cells were cultured to a confluence of about 80%, and the cells were collected after trypsinization, washed three times with PBS, and treated with TRIzol to extract RNA.

[0040] The reverse transcription kit uses the RevertAid First Strand cDNA Synthesis Kit, the reaction system is shown in Table 1, the concentration of Oligo(dT) primer is 100 μM; the reverse transcription reaction conditions include: Stage...

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Abstract

The invention provides a primer for quantitatively determining Twist-1 gene expression quantity in mesenchymal stem cells, a standard product and a detection method. The primer sequence is shown as SEQ ID No: 1 and SEQ ID No: 2. The detection method comprises the following steps of extracting total RNA of the mesenchymal stem cells; then performing inverse transcription to synthesize cDNA; using cDNA as a template; using the primer for PCR amplification; recovering and purifying amplification products for preparing plasmid standard products; drawing a standard curve; using cDNA of a sample tobe tested as a template; using the primer for fluorescent quantitative PCR reaction to obtain a Ct value of a sample to be tested; calculating the copy number of Twist-1 genes in the sample to be tested according to the standard curve. When the primer, the standard product and the detection method provided by the invention are used for performing repeated detection on the Twist-1 gene expression quantity in the mesenchymal stem cells, the repeated performance is good; the accuracy is high; the sensitivity is high; the detection time is short.

Description

technical field [0001] The invention belongs to the field of biological technology, and in particular relates to a primer, a standard product and a detection method for quantitative detection of Twist-1 gene expression in mesenchymal stem cells. Background technique [0002] Twist-1 is a transcription factor belonging to the basic helix-loop-helix protein family, which plays an important role in embryonic development, especially in the formation of mesoderm. Twist-1 gene is also involved in physiological and pathological activities such as tumor cell proliferation, apoptosis, epithelial-mesenchymal transition, and tumor cell drug resistance. [0003] Studies have pointed out that mutations in the Twist-1 gene can lead to symptoms such as dysphagia syndrome and radial hypoplasia; heterozygous Twist-1 mice often have craniofacial and limb deformities, and are prone to early death during embryonic development. As a key regulator of epithelial-mesenchymal transition, Twist-1 pl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6886C12Q2600/158C12Q2561/113C12Q2563/107C12Q2545/114
Inventor 刘宇张蓉谭遥黄晓霞
Owner CHENGDU ZHONGCHUANG QINGKE MEDICAL LAB CO LTD
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