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Reagent kit for quantitatively assessing long-term recurrence risks of breast cancer

A long-term recurrence and quantitative evaluation technology, applied in the direction of material excitation analysis, microbial measurement/inspection, fluorescence/phosphorescence, etc., can solve the problems that affect the development of detection and less attention, and achieve stable detection results, low detection cost, and easy operation. simple and fast effect

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

AI Technical Summary

Problems solved by technology

In foreign countries, "21 gene detection" only exists as a detection service, which seriously affects the clinical development of this detection. In addition, there is still relatively little attention in China. At present, this detection has not been carried out in our domestic clinics.

Method used

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  • Reagent kit for quantitatively assessing long-term recurrence risks of breast cancer
  • Reagent kit for quantitatively assessing long-term recurrence risks of breast cancer
  • Reagent kit for quantitatively assessing long-term recurrence risks of breast cancer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0106] Embodiment 1, the selection of clinical specimen and RNA extraction

[0107] Paraffin samples from the primary site were taken, and qualified tissue samples were screened after HE staining of tissue sections, and samples with less than 5% tumor cells were excluded.

[0108] Total RNA was extracted from 6 paraffin tissue slices cut into 10 μm thick by Beijing Huamei Shengke Biotechnology Co., Ltd. (Qiagen, product number: 74106).

[0109] Total RNA needs to be treated with DNase I to remove DNA impurities.

[0110] use PCR is for β-actin DNA (ABI, product number: 401846) to detect whether there is any DNA residue, if there is any, it needs to be treated with DNase I again. Quantification of total RNA.

Embodiment 2

[0111] Embodiment 2, reverse transcription

[0112] The specific primer working solution concentration of 21 genes is 10 μM, 0.4 μl of reverse primer solution (final concentration is 200nM) is mixed for each of the 21 genes, 2 μl of 10×RT-PCR buffer (10 is the dilution factor), and dNTP is mixed Solution 4 μl (final concentration is 500nM), RNase inhibitor 1 μl (10 units), reverse transcriptase 1 μl (4 units), RNA template extracted in Example 1 50ng, and the remaining volume was supplemented with water.

[0113] Mix the upper amplification system evenly and incubate at 37°C for 60 minutes.

Embodiment 3

[0114] Embodiment 3, taqman-real-time fluorescent quantitative PCR

[0115] The 21 genes were detected by fluorescent quantitative PCR, and one PCR reaction was performed for each gene. The reaction system was prepared as follows: 200 nM each of the forward and reverse specific primers, 2 μl of 10×PCR buffer, 1.6 μl (200 nM) of dNTP mixture, 0.1 μl (0.5 units) of DNA polymerase, and 100 nM of taqman fluorescent probe , 0.8 μl of the reverse transcription product in Example 2 (equivalent to the reverse transcription product of 2ng RNA template), and the remaining volume was supplemented by water.

[0116] The amplification reaction was performed at 95° C. for 15 minutes, 95° C. for 10-15 seconds, 60° C. for 10-15 seconds, and 72° C. for 10-15 seconds, for 30 cycles, and 72° C. for 7 minutes. After the amplification reaction, the Ct value of each gene was recorded, representing the expression level of each gene.

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Abstract

The invention relates to the functional genomic and gene expression detection technology and the analysis technology, and discloses a reagent kit for quantitatively assessing long-term recurrence risks of breast cancer. Particularly, a type of genes capable of being used for breast cancer metastasis and prognostic molecular classification is screened within a human functional genome expression profile range, the detection technology is created, and the reagent kit is prepared and applied to breast cancer metastasis and prognostic assessment for a patient. The reagent kit for quantitatively assessing long-term recurrence risks of breast cancer comprises 21 pairs of primers, 21 specific taqman fluorescent probes, 10XRT-PCR (reverse transcription-polymerase chain reaction) buffer solution, 2.5mM of dNTP (diethyl-nitrophenyl thiophosphate) mixed liquor, reverse transcriptase, DNA (deoxyribose nucleic acid) polymerase, 10XPCR buffer solution and RNA (ribonucleic acid) enzyme inhibitor. The reverse transcription PCR technology is combined with the taqman fluorescent quantitative PCR technology, reverse transcription primers, real-time PCR primers and the taqman fluorescent probes are self-designed and optimized, reverse transcription PCR reagent and taqman fluorescent quantitative PCR reagent are integrated to prepare the detection reagent kit, operation is simple and fast, detection results are more stable, and detection cost is lower.

Description

Technical field: [0001] The invention relates to functional genomics, gene expression detection technology and analysis technology. Specifically, it is to screen a class of genes that can be used for breast cancer metastasis and prognosis molecular typing within the range of human functional genome expression profiles, and establish detection technology, prepare kits, and apply to the metastasis and prognosis assessment of breast cancer patients. Background technique: [0002] Breast cancer is one of the malignant tumors that threaten women. In the past 20 years, we have gained a deeper understanding of the mechanism of tumorigenesis. The clinical practice of multidisciplinary comprehensive treatment centered on surgery and assisted by chemotherapy, radiotherapy, and endocrine therapy However, the diagnosis and treatment of breast cancer still rely heavily on traditional histopathology and immunohistochemical techniques, lacking the ability to accurately predict the recurren...

Claims

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Application Information

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