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Osteosarcoma diagnostic kit and application of CPE (carboxypeptidase E) gene to preparing osteosarcoma diagnostic kit

A diagnostic kit and gene technology, applied in the field of biomedicine, can solve problems such as inconsistent analysis results

Inactive Publication Date: 2014-09-24
杨祚璋
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, differences in different experimental platforms and samples lead to many inconsistencies in the analysis results of each gene chip.

Method used

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  • Osteosarcoma diagnostic kit and application of CPE (carboxypeptidase E) gene to preparing osteosarcoma diagnostic kit
  • Osteosarcoma diagnostic kit and application of CPE (carboxypeptidase E) gene to preparing osteosarcoma diagnostic kit
  • Osteosarcoma diagnostic kit and application of CPE (carboxypeptidase E) gene to preparing osteosarcoma diagnostic kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Screening for genes associated with osteosarcoma

[0044] 1.1 NCBI GEO (Gene Expression Omnibus) data retrieval

[0045] The GEO (Gene Expression Omnibus) database is developed and maintained by NCBI (National Center for Biotechnology Information). The GEO database is the largest database of gene expression data. SAGE (Serial Analysis of Gene Expression) data, SARST (Serial Analysis of Ribosomal Sequence Tags) data, MS (Mass Spectrometry) data, proteome data and next-generation high-throughput sequencing data (MPSS, massively parallel sequencing technology), etc.

[0046] a. Search keywords:

[0047] ("osteosarcoma"[MeSH Terms] OR osteosarcoma[All Fields])AND"Homo sapiens"[porgn]

[0048] b. Sample selection strategy in the study:

[0049] Limit the research type to "expression profiling by array" Data sets that meet the following criteria will be included in our research: ①The selected data set must be genome-wide expression mRNA microarray data; ②These da...

Embodiment 2

[0054] Embodiment 2QPCR verifies the relationship between candidate genes and osteosarcoma

[0055] Based on the results of Meta-analysis of osteosarcoma gene chip data, according to the size of the P value, we selected the CPE gene (Meta-analysis showed that the expression of CPE gene was up-regulated in osteosarcoma tissue compared with normal tissue) for verification. Collect 6 osteosarcoma tissue samples and 6 normal tissue samples at the same time, and use fluorescent real-time quantitative PCR to carry out classical molecular biology experiment verification (qRT-PCR). The specific operation steps are as follows:

[0056] (1) RNA extraction

[0057] After collecting the samples, freeze them in liquid nitrogen. After taking them out, put the tissue into a pre-cooled mortar for grinding. After the tissue sample is powdered:

[0058] ① Add Trizol and store at room temperature for 5 minutes;

[0059] ② Add 0.2ml of chloroform, vibrate the centrifuge tube vigorously, mix wel...

Embodiment 3

[0068] The preparation of embodiment 3 osteosarcoma diagnostic kits

[0069] According to the correlation between CPE gene and osteosarcoma, whether osteosarcoma occurs can be diagnosed by detecting the expression of CPE gene. Accordingly, the present invention provides a kit for diagnosing osteosarcoma based on detecting CPE gene expression. The diagnostic kit The components are as follows: SYBR Green polymerase chain reaction system; primer pair for amplifying CPE gene and GAPDH gene. The forward primer sequence for amplifying CPE gene is 5'-AGCATCCAGTGATAACATC-3', the reverse primer sequence is 5'-CTAGGCGGTCATTCTTAC-3'; the forward primer sequence for amplifying GAPDH is 5'-TTTAACTCTGGTAAAGTGGATAT-3', reverse The primer sequence is 5'-GGTGGAATCATATTGGAACA-3'. The SYBR Green polymerase chain reaction system includes PCR buffer, dNTPs, and SYBR Green fluorescent dye. PCR buffer composition: 25mM KCL, 2.5mM MgCL 2 , 200mM (NH 4 ) 2 SO 4 .

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Abstract

The invention discloses an application of a CPE (carboxypeptidase E) gene to preparing a kit for diagnosing osteosarcoma. 8 sets of satisfactory osteosarcoma cases and normal crowd genome mRNA (messenger RNA) expression chip data are taken into a research scope by utilizing NCBI (national center of biotechnology information) GEO (gene expression omnibus) data retrieval; by carrying out Meta analysis onto the chip data, the CPE gene is screened as a difference significant gene; compared with a normal structure, expression of the CPE gene in the osteosarcoma tissue is regulated up, and the conclusion is verified by using a fluorescent real-time quantification PCR (polymerase chain reaction) method. The invention further discloses a kit for diagnosing osteosarcoma and a method for diagnosing osteosarcoma by utilizing the kit. According to the application disclosed by the invention, on one hand, a novel thought is provided for researching a molecular mechanism of the osteosarcoma, and on the other hand, a more flexible diagnostic kit is provided for diagnosing osteosarcoma at an early stage.

Description

technical field [0001] The invention belongs to the field of biomedicine and relates to an osteosarcoma diagnostic kit and the application of CPE gene in preparing the osteosarcoma diagnostic kit. Background technique [0002] The earliest and most widely used in transcriptome research is the gene chip technology. First, RNA is extracted from the sample to be tested, and it is reverse-transcribed into cDNA using fluorescently labeled nucleotides. The labeled nucleotide sequence can be compared with The probe hybridization on the specific site of the gene chip, and the cell gene expression information can be obtained by detecting the hybridization signal. Gene chip technology has become a very stable and credible experimental technology, and a large amount of transcriptome data published so far is mainly generated by gene chip technology. [0003] Osteosarcoma is a malignant tumor derived from mesenchymal tissue. Its main pathogenic feature is that the proliferating tumor ce...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6851C12Q1/6886C12Q2600/158C12Q2531/113C12Q2563/107C12Q2561/113
Inventor 杨祚璋杨义豪李东奇陈彦锦刘雪峰
Owner 杨祚璋
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