Q-PCR detection method for colorectal cancer marker DCD

A detection method, a colorectal cancer technology, is applied in the field of Q-PCR detection of the colorectal cancer marker DCD, which can solve problems such as the unsystematic establishment of the detection method, and achieve the effects of preventing secondary pollution, strong specificity, and high sensitivity

Pending Publication Date: 2021-05-25
广州医科大学附属中医医院
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  • Abstract
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  • Application Information

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Problems solved by technology

However, the detection method of DCD in colorectal cancer cells has not been systematically established.

Method used

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  • Q-PCR detection method for colorectal cancer marker DCD
  • Q-PCR detection method for colorectal cancer marker DCD
  • Q-PCR detection method for colorectal cancer marker DCD

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1～2

[0037] Reagents used in Examples 1-2

[0038]

[0039]

Embodiment 1

[0043] A Q-PCR detection method for colorectal cancer marker DCD, characterized in that it comprises the following steps: 1) selecting a normal human epithelial cell (HIEC) and five human colon cancer cells as cell samples; the five human colon cancer cells The cancer cells were SW620 cells, LoVo cells, HT-29 cells, HCT-116 cells and SW480 cells;

[0044]The RNA extraction method of the cell sample is as follows: After the cell sample has been resuscitated and cultured, add 1mL TRIzol RNA isolation reagent and let it stand at room temperature; then add 0.2mL chloroform, shake, and centrifuge at 4°C and 12000g for 15min; absorb the upper aqueous phase, transfer To another centrifuge tube, add 0.5mL isopropanol, mix well, let stand at 37°C for 10min, then centrifuge at 4°C, 12000g for 10min, discard the supernatant; continue to add 1mL 75% ethanol, shake Finally, centrifuge at 4°C and 7500g for 5min, discard the supernatant; finally dry naturally at 37°C, add 20 μL DEPC water to...

Embodiment 2

[0063] A Q-PCR detection method for colorectal cancer marker DCD, characterized in that it comprises the following steps:

[0064] 1) Select five groups of colorectal cancer tissues (T) and five groups of paracancerous tissues (N is normal tissue) as tissue samples;

[0065] The RNA extraction method of tissue samples is as follows: cut 0.1g of tissue samples, add 1mL TRIzol RNA isolation reagent, and let it stand at room temperature; then add 0.2mL chloroform, after shaking, centrifuge at 4°C and 12000g for 15min; absorb the upper aqueous phase, transfer To another centrifuge tube, add 0.5mL isopropanol, mix well, let stand at 37°C for 10min, then centrifuge at 4°C, 12000g for 10min, discard the supernatant; continue to add 1mL 75% ethanol, shake Finally, centrifuge at 4°C and 7500g for 5min, discard the supernatant; finally dry naturally at 37°C, add 20 μL of DEPC water to obtain the RNA of the tissue sample.

[0066] 3) Reverse transcription: Prepare 20 μL of the first-str...

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Abstract

The invention relates to the technical field of in-vitro detection, in particular to a Q-PCR detection method of a colorectal cancer marker DCD. The method comprises the steps of RNA extraction of a sample, reverse transcription and Q-PCR amplification. The DCD is used as a colorectal cancer candidate diagnosis index and treatment target, RNA of cells or tissues is firstly extracted, an internal reference primer and a DCD gene primer are set to perform reverse transcription on the RNA to obtain two groups of different amplification products, Q-PCR reaction is performed respectively to obtain melting curves and amplification curves of the two groups of amplification products respectively, and the expression level of the DCD gene is calculated; then, theexpression level of the DCD geneis compared with the mRNA concentration of the DCD in the normal tissue or cell, and if the mRNA concentration of the DCD in the tested tissue or cell is more than one time higher than that of the normal tissue or cell, the tested tissue or cell can be considered to be the colorectal cancer tissue or colorectal cancer cell, so that early diagnosis of the colorectal cancer is facilitated.

Description

technical field [0001] The invention relates to the technical field of in vitro detection, in particular to a Q-PCR detection method for a colorectal cancer marker DCD. Background technique [0002] DCD (Dermcidin) is a natural active peptide isolated from human sweat by German scientists Schittek et al. in 2001. DCD was initially thought to be specifically expressed in sweat glands, expressed in sweat glands and secreted into sweat. antibacterial activity. Later studies found that DCD is also expressed in certain cancers, including melanoma, skin tumors, breast, prostate, pancreas, lung and hepatocellular carcinoma. DCD is likely to be a potential oncogenic factor and could serve as a candidate therapeutic target for the treatment of these cancers. [0003] Experimental studies have confirmed that DCD is highly expressed in colorectal cancer, which ranks third in the incidence of tumors in the world, and can become a diagnostic index and therapeutic target for colorectal ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6886C12Q2600/158C12Q2561/113C12Q2563/107C12Q2545/114
Inventor 邱芳华李秋明龙华婧刘道利
Owner 广州医科大学附属中医医院
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