Kit for quantitative evaluation for long-term recurrent risk of colorectal cancer

A technology for colorectal cancer and long-term recurrence, applied in material excitation analysis, fluorescence/phosphorescence, etc., can solve problems such as failure to predict curative effect

Inactive Publication Date: 2012-07-18
苏州科贝生物技术有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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However, the model based on the efficacy-related

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  • Kit for quantitative evaluation for long-term recurrent risk of colorectal cancer
  • Kit for quantitative evaluation for long-term recurrent risk of colorectal cancer
  • Kit for quantitative evaluation for long-term recurrent risk of colorectal cancer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0242] Example 1. Selection of clinical specimens and RNA extraction

[0243] 1. Take a paraffin sample from the primary position and screen the qualified tissue samples after HE staining of the tissue section, and exclude samples with less than 5% of tumor cells.

[0244] 2. Take 6 pieces of paraffin tissue pieces cut into 10 microns thick, and extract total RNA from them using RNeasy Mini Kit (Qiagen, Item No. 74106).

[0245] 3. Treat total RNA with DNase I to remove DNA impurities.

[0246] 4. Use PCR assay for β-actin DNA (ABI, catalog number: 401846) detects DNA residues. Quantify total RNA.

Embodiment 2

[0247] Example 2. Reverse transcription amplification technology (RT-PCR) to prepare cDNA

[0248] Prepare cDNA by reverse transcription amplification: prepare the mixed solution for reverse transcription amplification: total RNA template 50ng, 18 reverse primer working solutions each take 0.4μl (final concentration is 200nM), 10×RT-PCR buffer 2μl, 4 μl of dNTP mixture, 1 μl of RNase inhibitor, 1 μl of reverse transcriptase, RNA extracted in Example 1, and the remaining volume made up with water. Mix the upper amplification system uniformly and incubate at 37°C for 60 minutes to obtain cDNA.

Embodiment 3

[0249] Example 3 Taqman real-time PCR (Taqman real-time PCR)

[0250] Real-time fluorescence quantitative PCR amplification was performed on the above-mentioned amplified products. 18 genes were separately performed. Each PCR reaction used a pair of specific primers and a specific Taqman fluorescent probe for detecting the amplified products of the primers.

[0251] 1. Each PCR reaction system is prepared as follows: two specific primers for forward and reverse are 200nM, 10×PCR buffer 2μl, dNTP mixture 1.6μl (200nM), DNA polymerase 0.1μl, Taqman fluorescent probe 100nM , 0.8μl of the reverse transcription product in Example 2 (equivalent to the reverse transcription product of 2ng RNA template), and the remaining volume is made up of water.

[0252] 2. The amplification reaction is carried out at 95°C for 15 minutes, 95°C for 10-15 seconds, 60°C for 10-15 seconds, 72°C for 10-15 seconds, for 30 cycles, 72°C for 7 minutes. After the amplification reaction, record the Ct value of eac...

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Abstract

The invention relates to functional genomics and a gene expression detection and analysis technology, in particular to a kit for quantitative evaluation for long-term recurrent risk of colorectal cancer. The kit is characterized by consisting of 18 pairs of primers, 18 specific taqman fluorescent probes, 10* reverse transcription-polymerase chain reaction (RT-PCR) buffer solution, deoxyribonucleoside triphosphate (dNTP) mixed liquid, reverse transcriptase, deoxyribose nucleic acid (DNA) polymerase, 10* PCR buffer solution and ribonucleic acid (RNA) enzyme inhibitor. By adopting the self-designed and optimized RT primers and integrating a RT-PCR technology and a taqman real-time fluorescent quantitative PCR technology, the kit is simple and rapid in operation, more stable in diction results and lower in detection cost.

Description

Technical field [0001] The invention relates to functional genomics and gene expression detection and analysis technology. Specifically, it is to screen a class of genes that can be used for colorectal cancer metastasis and prognostic molecular typing within the range of human functional genome expression profiles, and establish detection technologies to prepare kits for metastasis and prognosis evaluation of colorectal cancer patients . Background technique [0002] Colorectal cancer is one of the most common malignant tumors. In the past 20 years, the incidence of colorectal cancer has been increasing in most countries in the world. The increasing trend of the incidence of colorectal cancer in China is also very obvious. The age of onset is mostly 40-60 years old, and the peak is around 50 years old. However, colorectal cancer patients under 30 years old are not uncommon. [0003] Colorectal cancer has an insidious onset, and the early symptoms are not obvious. Many patients a...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N21/64
Inventor 侯青姬云张泓
Owner 苏州科贝生物技术有限公司
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