Application of PLEKHA5 in preparation of tumor diagnosis reagent

A preparation and kit technology, applied in the field of preparation of osteosarcoma diagnostic reagents, can solve the problems of high mortality, confusion of trauma, great harm, etc., and achieve the effects of good repeatability, clear results and rapid response

Active Publication Date: 2015-07-01
北京鸿志合创科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Osteosarcoma has no obvious clinical symptoms at the onset, and is easily confused with trauma, and lung met

Method used

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  • Application of PLEKHA5 in preparation of tumor diagnosis reagent
  • Application of PLEKHA5 in preparation of tumor diagnosis reagent
  • Application of PLEKHA5 in preparation of tumor diagnosis reagent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 High-throughput sequencing and analysis

[0033] Collect 5 cases of osteosarcoma samples and 3 cases of normal control tissues for RNA extraction. After RNA extraction, agarose gel electrophoresis can be used to preliminarily determine whether the quality of the extracted RNA samples is qualified or not, and whether they can be used for further transcription group analysis. Then, the extraction of RNA samples was detected by NanoDrop1000 spectrophotometer, and the sample requirements for RNA-seq sequencing: OD260 / OD280 was 1.8-2.2.

[0034] The sequencing platform is the HiSeq 2500 high-throughput sequencing platform of Illumina, which performs high-throughput transcriptome deep sequencing. After sequencing, we use Fast-QC (http: / / www.bioinformatics.babraham.ac.uk / projects / fastqc / ) The software evaluates the quality of sequencing data as a whole, including base quality value distribution, quality value position distribution, GC content, PCR duplication conten...

Embodiment 2

[0035] Example 2 Osteosarcoma tissue and normal tissue PLEKHA5 gene expression

[0036] 1. Materials and methods

[0037] 1. Materials

[0038] 65 cases of osteosarcoma tissues and 25 cases of normal tissues were collected, grouped and numbered.

[0039] 2. Method

[0040] 2.1 Extraction of total RNA from osteosarcoma tissue and normal tissue

[0041] use Reagent (invitrogen, Cat. No. 15596-018) was used to extract sample RNA, and the experimental operation was carried out according to the product manual. The specific operation was as follows:

[0042] 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:

[0043] ① Add Trizol and store at room temperature for 5 minutes;

[0044] ② Add 0.2ml of chloroform, vibrate the centrifuge tube vigorously, mix well, and place it at room temperature for 5-10 minutes;

[0045] ③ 12000rpm high-speed centrifugatio...

Embodiment 3

[0071] Example 3 Culture of osteosarcoma cell line MG-63

[0072] 1. Materials

[0073] (1) Source of specimens

[0074] The human osteosarcoma cell line MG-63 was purchased from the Shanghai Cell Institute of the Chinese Academy of Sciences.

[0075] (2) Main reagents

[0076] DMEM / HIGH Glucose (1×) (Thermo Fisher Biochemicals (Beijing) Co., Ltd.)

[0077] (3) Main solution

[0078] 1. Cell culture medium

[0079] DMEM medium + 10% standard fetal bovine serum.

[0080] 2. PBS (balanced salt solution)

[0081] Dissolve 8g NaCl, 0.25g KCl, 1.44g Na in 800ml distilled water 2 HPO 4 and 0.24g KH 2 PO 4 Adjust the pH value of the solution to 7.4 with HCl, add water to make up to 1 L, autoclave, and store at room temperature.

[0082] 3. 0.25% trypsin digestion solution

[0083] Add 0.25g of trypsin to 100ml of deionized water, filter and sterilize it, and distribute it for use.

[0084] 2. Experimental method

[0085] (1) Cell culture

[0086] 1. Cell passage

[0087...

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Abstract

The invention relates to application of PLEKHA5 in preparation of a tumor diagnosis reagent, in particular to a new application of the PLEKHA5 in preparation of an osteosarcoma diagnosis reagent. The inventor analyzes and screens genes by virtue of a bioinformatics method on the basis of a high-throughput sequencing result, and picks out a candidate gene PLEKHA5; furthermore, a molecular cell biology experiment proves that the PLEKHA5 and osteosarcoma have good correlation; and the PLEKHA5 can be used for preparing an auxiliary diagnosis reagent for osteosarcoma, and has important clinical application value.

Description

technical field [0001] The present invention relates to the field of biomedicine, and the application of PLEKHA5 in the preparation of tumor diagnostic reagents. Specifically, the present invention relates to the new application of PLEKHA5 in the preparation of osteosarcoma diagnostic reagents. Background technique [0002] PLEKHA5 is a member of the PLEKHA((pleckstrin homology domain-containing protein family A) family, and there are very few studies on this gene. Kenichiro et al. (Identification and characterization of splicing variants of PLEKHA5(Plekha5) during brain development, Gene, Volume 492 , Issue 1, 15January 2012, 270-275) after studying the gene, it was found that its mRNA has two expression forms, and the longer mRNA chain (L-PLEKHA5) has 32 exons, encoding 1282 amino acids. Expression is higher in the brain tissue, and shorter (S-PLEKHA5) has 26 exons, encodes 1116 amino acids, and is expressed in various parts of the body. The protein encoded by these two fo...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61K31/7088C12Q1/68A61P35/00
Inventor 杨承刚边洋孙耀兰任静
Owner 北京鸿志合创科技有限公司
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