Application of SRGN gene in serving as serum marker for detecting triple-negative breast cancer

A triple-negative breast cancer, marker technology, applied in the field of biomedicine, can solve problems that have not been studied and reported

Inactive Publication Date: 2016-05-18
CANCER CENT OF GUANGZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, there are no relevant studies and repor...

Method used

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  • Application of SRGN gene in serving as serum marker for detecting triple-negative breast cancer
  • Application of SRGN gene in serving as serum marker for detecting triple-negative breast cancer
  • Application of SRGN gene in serving as serum marker for detecting triple-negative breast cancer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Real-time quantitative PCR analysis of SRGN expression in breast cancer cell lines

[0040] 1. Prepare cDNA

[0041] Take various cultured cells that grow to the logarithmic phase, and extract total RNA by TRIZOL method. After DNaseI digestion, RNA was measured.

[0042] Using oligdT as the primer, the same amount of RNA was reverse transcribed into cDNA according to the Fermentas ReverseTranscriptionSystem program.

[0043] 2. PCR detection

[0044] Using cDNA as a template, follow the SYBGreen operating instructions and use the following primers for real-time quantitative PCR amplification:

[0045] (1) Primer of SRGN gene coding region (amplified fragment is 100bp):

[0046] Forward primer SRGN-F (shown in SEQ ID NO.3):

[0047] 5'-GCTACTCAAATGCAGTCGGC-3';

[0048] Backward primer SRGN-R (shown in SEQ ID NO. 4):

[0049] 5'-CCATTGGTACCTGGCTCTCC-3'.

[0050] (2) The primer amplified fragment of GAPDH gene coding region is 156bp:

[0051] Forward primer GAPDH-F (shown in SE...

Embodiment 2

[0058] Example 2 Westenblot analysis of SRGN expression in breast cancer cell lines

[0059] 1. Westenblot

[0060] Take the cultured cells that have grown to the logarithmic phase, use fresh cell lysate containing protease inhibitors to lyse the cells to extract total protein. The protein concentration was determined by the Bradford method of Bio-Rad. Add 40μg protein to each lane to calculate the cell protein volume, add 6×SDS loading buffer at a ratio of 1:5 to dissolve it, denature it at 100°C for 5min, immediately place it on ice for 5min, and centrifuge briefly. After loading the sample, perform electrophoresis. First, electrophoresis at 80V until the bromophenol blue dye enters the separation gel, and then adjust the voltage to 120V to continue electrophoresis until the bromophenol blue reaches the bottom of the gel. The Bio-Rad electrophoresis instrument conducts electrotransfer of protein at 120mV constant pressure for 90min, so that the separated protein is transferred...

Embodiment 3

[0063] Example 3 Enzyme-linked immunoassay (ELISA) analysis of SRGN expression in the supernatant of breast cancer cell lines

[0064] 1. Prepare the cell culture supernatant: After digesting the target cells into a single cell suspension and counting, add 1×10 6 Cells / well were seeded in a 6-well plate with a final culture volume of 2ml. After 48 hours of culture, the culture supernatant was collected, and the supernatant was collected after centrifugation at 2000 rpm / min for ELISA experiments.

[0065] 2. Brief steps of ELISA experiment:

[0066] (1) All reagents in the kit are slowly balanced to room temperature before use to avoid rapid dissolution and protein degradation.

[0067] (2) Prepare standard diluent: add 1ml standard diluent to the standard, let it stand at room temperature for 10 minutes, and rub it repeatedly to promote dissolution. At this time, the concentration is 20ng / ml. Then prepare 7 standard dilution EP tubes, add 250μl standard dilution solution to each EP t...

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Abstract

The invention discloses application of an SRGN gene and polypeptide thereof in serving as a detection marker for breast cancer molecular typing, in particular to application of the SRGN gene and the polypeptide thereof in serving as a serum marker for detecting a triple-negative breast cancer or in preparation of a detection reagent for the triple-negative breast cancer, and provides a novel breast cancer serum marker, that is, the SRGN gene and the polypeptide thereof. It is proved that expression of the SRGN gene in breast cancer serum is enhanced, and expression of the SRGN gene in serum of a triple-negative breast cancer patient is mainly and significantly increased compared with expression of the SRGN gene in serum of a breast cancer patient with the other molecular types. Accordingly, not only is a new target supplied to breast cancer serology detection, but also new data and a quick serology detection method are supplied to breast cancer molecular typing; a new chemical entity is designed at the gene level and the protein level by taking the gene as the target, breast cancer molecular typing can be quickly, conveniently and effectively performed, and therefore the breast cancer treatment effect is improved.

Description

technical field [0001] The invention belongs to the technical field of biomedicine. More specifically, it relates to the application of SRGN gene as a serum marker for detecting triple-negative breast cancer. Background technique [0002] Breast cancer is one of the malignant tumors with the highest incidence rate and the most serious harm in women. In recent years, with the update of treatment methods, improvement of treatment schemes and diversification of treatment methods, the clinical treatment effect of breast cancer has been greatly improved. However, there are still about 500,000 people in the world who die of breast cancer every year. The reason is that breast cancer is a highly heterogeneous disease at the molecular level. The molecular genetic changes of tumors with the same clinical and histopathological morphology are not necessarily similar. These factors affect their treatment and prognosis. Future directions in cancer treatment. [0003] Tumor molecular c...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N33/68G01N33/574
CPCC12Q1/6886C12Q2600/112C12Q2600/158G01N33/57415G01N33/57484G01N33/68G01N2333/47
Inventor 贺智敏张志杰罗凯邓印根郑国沛谷依学尹江邱霓
Owner CANCER CENT OF GUANGZHOU MEDICAL UNIV
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