Detection kit for SDPR gene expression and/or SDPR gene methylation level and application

A technology for gene expression level and detection reagents, which is applied in the determination/examination of microorganisms, DNA/RNA fragments, measurement devices, etc. effect of value

Pending Publication Date: 2020-09-08
THE FIRST AFFILIATED HOSPITAL OF CHONGQING MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, the methylation status, biological function and clinical ...

Method used

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  • Detection kit for SDPR gene expression and/or SDPR gene methylation level and application
  • Detection kit for SDPR gene expression and/or SDPR gene methylation level and application
  • Detection kit for SDPR gene expression and/or SDPR gene methylation level and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Expression of SDPR in non-small cell lung cancer

[0049] 1. Extraction of cellular RNA

[0050] Lung cancer cells H1299, H2122, H358, H446, H460 and A549 cells were placed in 1640 complete medium containing 10% fetal bovine serum at 37°C and 5% CO 2 Cultured in an incubator, and when the cell fusion rate reached 70% to 80%, it was digested with 0.25% trypsin and passaged.

[0051] Rinse the cells in the culture flask 2-3 times with PBS, drain the residual water, add 1mL Trizol to the flask, place on a shaker for 5min, collect the cells and lysate in a clean EP tube. Add 200 μL of chloroform, shake vigorously for 15 s, mix thoroughly and let stand at room temperature for 3 min, then centrifuge in a centrifuge at 4° C. (12000 rpm / min×15 min). Pipette the supernatant into a new EP tube, add 0.5mL isopropanol, mix upside down and place it for 10min, then centrifuge at 4°C (12000rpm / min×15min), a white precipitate can be seen at the bottom of the tube. Discard ...

Embodiment 2

[0077] Example 2 Methylation detection of SDPR

[0078] 1. DNA bisulfite modification

[0079] Prepare CT Conversion Reagent mixture: M-Dilution Buffer 300μL, M-DissolvingBuffer 50μL, DEPC H 2 O 900 μL. Add 24mL of 100% ethanol to M-Wash Buffer for later use.

[0080] Add 20 μL DNA to 130 μL CT Conversion Reagent, mix well and incubate. Reaction conditions: 98°C for 10min, 64°C for 2.5h, 4°C for 20h. Then transfer the liquid to the spin column, add 600μL M-Binding Buffer, mix well and centrifuge (12000rpm / 30s), discard the supernatant. Add 200μL M-Wash Buffer, centrifuge (12000rpm / 30s), and discard the supernatant. Add M-DesμLphonation Buffer, let stand at room temperature for 20min, centrifuge (12000rpm / 30s), and discard the supernatant. Add 200μL M-Wash Buffer, centrifuge (12000rpm / 30s), and discard the supernatant. Set the spin column into a new enzyme-free Ep tube, add 10 μL M-Elution Buffer, centrifuge (12000 rpm / 30s), collect the filtrate, and store it at -20 °C. ...

Embodiment 3

[0092] Example 3 The effect of SDPR on lung cancer

[0093]1. CCK8 detection of cell proliferation ability

[0094] Construct a cell line stably expressing and knocking out SDPR, and use RT-PCR and Western verification (such as Figure 4 As shown in A), SDPR was successfully overexpressed and knocked out in H1299 and A549 cells, respectively. Collect cells and resuspend with culture medium, adjust cell concentration to 2×10 4 Cells / mL, 100 μL of cell suspension per well was placed in a 96-well plate (4 replicate wells for each group), placed in an incubator for 24, 48, and 72 hours of culture, and then CCK8 was detected. Specific steps: discard the culture medium of the measured wells, then add 110 μL of mixed solution (1 mL of medium + 100 μL of CCK8) to each well in the dark, continue to cultivate for 2 hours, and then measure the absorbance value of each well at a wavelength of 450 nm on a microplate reader, and the results (such as Figure 4 As shown in B), overexpressi...

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Abstract

The invention discloses application of an SDPR gene expression level detection reagent and/or an SDPR gene methylation level detection reagent in preparation of a kit used for diagnosing whether a subject suffers from lung cancer or not, or used for predicting whether the subject has a lung cancer risk or not, or used for judging whether a lung cancer patient is sensitive to chemotherapeutic drugsor not, or used for judging whether the lung cancer patient has good prognosis or not, and belongs to the field of molecular diagnosis. The invention provides a new clinical marker for early diagnosis and treatment of lung cancer, and has huge clinical value.

Description

technical field [0001] The invention belongs to the field of molecular diagnosis, and in particular relates to a detection kit and application of SDPR gene expression and / or SDPR gene methylation level. Background technique [0002] Lung cancer is one of the malignant tumors with the fastest-growing morbidity and mortality in the world and the greatest threat to the health and life of the population. The degree of malignancy of the tumor is very high, the prognosis is the worst among all tumor types, and its 5-year survival rate is only 10%–20%. Despite advances in the treatment of non-small cell lung cancer (NSCLC) over the past few years, its overall survival remains poor. One of the major problems is chemotherapy resistance. The treatment of non-small cell lung cancer (NSCLC) includes surgery, radiotherapy, chemotherapy and molecular targeted therapy. At present, for resectable lung cancer, surgery is still the most important treatment, and radiotherapy and chemotherap...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12N15/11G01N33/574
CPCC12Q1/6886G01N33/57423C12Q2600/154C12Q2600/158C12Q2600/118
Inventor 向廷秀郭述良彭明昱彭溦雁唐俊邱祝
Owner THE FIRST AFFILIATED HOSPITAL OF CHONGQING MEDICAL UNIVERSITY
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