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A group of internal reference genes and their screening methods for real-time quantitative pcr relative quantification applied to hepatocellular carcinoma

An internal reference gene, real-time quantitative technology

Active Publication Date: 2021-02-19
上海市松江区中心医院
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The stability of the commonly used ACTB ranks third and can be used as a traditional alternative internal reference, while GAPDH, HPRT1 and TUBB cannot meet the requirements for use as internal reference genes in HCC cell lines in terms of expression stability

Method used

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  • A group of internal reference genes and their screening methods for real-time quantitative pcr relative quantification applied to hepatocellular carcinoma
  • A group of internal reference genes and their screening methods for real-time quantitative pcr relative quantification applied to hepatocellular carcinoma
  • A group of internal reference genes and their screening methods for real-time quantitative pcr relative quantification applied to hepatocellular carcinoma

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Experimental program
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Effect test

Embodiment 1

[0026]A total of 48 Affymetrix HG U133 Plus 2.0 Array genome-wide expression profile chips were collected from 10 HCC cell lines (lines), as shown in Tables 1 and 2. The two chip data sets of MHCC-97L, MHCC-97H, HCCLM3, HCCLM6 and Hep3B are independently developed and produced, Huh-7, PLC / PRF / 5, SNU-398, SNU-449, SNU-475 and Hep3B The data of the 3 chips comes from the public data sets ArrayExpess and GEO. Since the number of parallel measurement chips in each cell is different, in order to avoid weight differences, the data is screened by cell type as a unit. The expression levels of genes in 10 liver cancer cell lines are all represented by the arithmetic mean of parallel measurement of MAS 5.0 signal values. The screening criteria are mainly based on the coefficient of variation CV of gene expression in 10 types of liver cancer cellsAnd to control the range of expression between any two cells must not be too high MFC = Max (Ii) / Min(Ii)iRepresents the gene expression in each cell ...

Embodiment 2

[0036]Including the following 9 hepatocellular carcinoma cell lines Huh-7, Hep3B, PLC / PRF / 5, MHCC-97L, MHCC-97H, HCCLM3, SNU-398, SNU-449, SNU-475. Huh-7, Hep3B, PLC / PRF / 5, MHCC-97L, MHCC-97H, HCCLM3 were passaged and cultured in DMEM medium with 10% fetal calf serum. SNU-398, SNU-449, and SNU-475 were subcultured and cultured in RPMI 1640 medium with 10% fetal bovine serum. Collect 5×10 for each cell line6Cells, use Trizol reagent to extract total RNA in cells according to the operating method of the instructions, and then perform DNase I treatment and cDNA reverse transcription.

[0037]Primers for 16 genes were designed by PerlPrimer v1.1.16, including AIMP1, ANP32B, BCL2L13, CNPY3, CUGBP1, ENY2, HNRNPC, RPL22, SEC61B, SFRS4, TFG, TMED2, TROVE2, UBE2D3, UBE2V2 and YWHAB. The primers of TSFM and UBE2B are designed by NCBI primer BLAST. The primers of UBE2N and GAPDH are designed by PrimerPremier 5.0. ACTB amplification primers are taken from literature. The primers for HPRT1 (1645189...

Embodiment 3

[0041]Through the analysis of gene expression stability and expression intensity at the gene and probe levels of 48 genome-wide expression profile chip data of 10 cell lines, 19 candidate genes were screened out for subsequent verification. The specific results are as followsfigure 1 Shown.

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Abstract

The invention relates to the field of gene technology, in particular to a group of real-time quantification PCR (Polymerase Chain Reaction) relative quantification reference genes applied to hepatocellular carcinoma and a screening method thereof. TFG and SFRS4 can be used as relatively stable reference genes in the study of hepatocellular carcinoma cell lines. Through assessment on the expressionstability of common reference genes, ACTB can be applied as an internal reference, and GAPDH, HPRT1 and TUBB are unsuitable to serve as reference genes associated with hepatocellular carcinoma researches.

Description

Technical field[0001]The present invention relates to the field of gene technology, in particular to a set of real-time quantitative PCR relative quantitative internal reference genes applied to hepatocellular carcinoma and a screening method thereof.Background technique[0002]Gene expression detection is an important method of molecular biology research. Real-time quantitative PCR (qRT-PCR), as a research strategy that can accurately quantify gene expression levels, has been widely used in recent years. qRT-PCR can be divided into absolute and relative quantification. The relative quantification method is more widely used due to its high sensitivity, good repeatability, easy operation and reliable results. Based on 2-ΔΔCt The calculation strategy of is currently the most common relative quantitative method used to obtain the expression level difference between two transcripts. This method needs to use the internal reference gene that is basically constant in the expression system as...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6886C12Q1/686C12N15/11C12N15/10G16B20/20
Inventor 钟凡刘阳徐萍王静赖跃兴杨志文
Owner 上海市松江区中心医院
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