Marker for cancer detection and application thereof

A cancer detection and marker technology, applied in the field of molecular identification of cancer detection, can solve the problem of lack of molecular markers in cancer, and achieve the effect of high accuracy and high reference value

Inactive Publication Date: 2013-04-17
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008]3) Certain types of cancer lack corresponding molecular markers
[0009]4) So far there is no single cancer molecular marker that can detect all types of cancer

Method used

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  • Marker for cancer detection and application thereof
  • Marker for cancer detection and application thereof
  • Marker for cancer detection and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 RT-PCR detection of testicular seminoma

[0045] 1. Total RNA extraction

[0046] 1) Immediately take 50 mg of fresh tumor tissue, place it in a mortar, carefully pour an appropriate amount of liquid nitrogen, and grind it quickly until the tissue becomes white powder.

[0047] 2) Add 1 mL of Trizol (purchased from Invitrogen), mix well, and lyse at room temperature for 5 minutes.

[0048] 3) Add 0.2mL chloroform, mix well, centrifuge at 12000rpm for 15 minutes, take the upper aqueous phase into a new centrifuge tube.

[0049] 4) Add 0.5mL isopropanol, mix well, centrifuge at 12000rpm for 15 minutes, discard the supernatant, the precipitate is RNA.

[0050] 5) Wash with freshly prepared 75% ethanol, centrifuge at 12,000 rpm for 15 minutes, and discard the supernatant.

[0051] 6) Add 20-25 μL of deionized water, and measure the OD to calculate the RNA concentration after the precipitate is dissolved.

[0052] 2. Preparation of cDNA template. See Table 1 f...

Embodiment 2

[0066] Example 2 Detection of various types of tumors by Western blot

[0067] 1. Extraction of protein

[0068] Soak the whole set of homogenizer in acid for more than 4 hours, wash and dry, pack with tin foil, and bake at 180°C for 2 hours. Take 100 mg of fresh tissue, add PBS, grind the tissue with a homogenizer, break the cells with an ultrasonic instrument, add the lysate, boil for 3 minutes, place on ice for 5 minutes, centrifuge at 5000rpm for 5 minutes, and take the supernatant for loading. Lysis buffer configuration, 50mmol / L Tris Base, 100mmol / L dithiothreitol, 2% (m / v) SDS, 0.1% bromophenol blue, 10% (v / v) glycerol.

[0069] 2. Western blot

[0070] 1) In the experiment, we used the SDS-PAGE gel separation system with a concentration of 15%, and the preparation methods are shown in Table 4 and Table 5. Electrophoresis buffer configuration, 25mmol / L Tris Base, 250mmol / L glutamic acid (pH8.3), 0.1% (m / v) SDS.

[0071] components Volume (mL) water ...

Embodiment 3

[0079] Example 3 Immunofluorescence detection of squamous cell carcinoma of the lung

[0080] 1. Frozen section

[0081] Pre-cool the cryostat to -20°C to -18°C. Choose the most suitable temperature according to the nature of different tissues. Serial sections were made with a thickness of 7 microns.

[0082] 2. Immunofluorescence

[0083] 1) Place frozen sections in pre-cooled methanol and fix at 4°C for 20 minutes. After fixation, wash three times with PBS at room temperature, 10 minutes each time. PBS configuration, 8 g NaCl, 0.2 g KCl, 1.44 g Na 2 HPO 4 , 0.24 g KH 2 PO 4 , adjust the pH to 7.4 with HCl, add water to 1 liter, and autoclave for 20 minutes.

[0084] 2) Put in 0.5%v / v TX-100, penetrate at room temperature for 20 minutes. Wash three times with PBS, 10 minutes each time.

[0085] 3) Add normal goat serum blocking solution and block for 1 hour at room temperature.

[0086] 4) join Nurim Primary antibody (generally the concentration is 1:100 dissolve...

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Abstract

The invention discloses a marker for cancer detection and an application thereof. The marker is mRNA of a Nurim gene or an expression product thereof; and a reagent comprises a reagent for specifically detecting the mRNA of the Nurim gene or a reagent for specifically detecting the expression product of the mRNA of the Nurim gene. Through RT-PCR detection of different transcripts or immunoblots of the Nurim or immunofluorescence assay of protein expression of the Nurim, whether a patient suffers a cancer or not can be diagnosed. The marker for the cancer detection, disclosed by the invention, is general and capable of detecting a plurality of different malignant tumors; meanwhile, according to the quantity of the mRNAs or the expression products, the tumor progression degree can be reflected; through the adoption of the molecular marker or the detecting reagent disclosed by the invention, the cancer can be conveniently and rapidly diagnosed with high accuracy; and the marker for the cancer detection, disclosed by the invention, has higher reference values to clinical diagnosis.

Description

technical field [0001] The invention relates to a molecular marker and technical method for cancer detection, and has broad application prospects in the fields of medical genetics and clinical diagnosis. Background technique [0002] 1. Molecular markers of cancer [0003] Human normal cells go through a series of gene mutations, and eventually get rid of the normal growth regulation of cells and become cancerous. These mutations involve chromosomal translocation, gene amplification, gene deletion, point mutation, etc. Cancer histogenetics are altered, ultimately leading to differential expression of certain gene mRNAs and proteins in cancer individuals. Therefore, although the functions of these genetic changes are not clear, we can still obtain biological information of cancer tissues through differences in gene mRNA and protein expression levels. [0004] Cancer molecular markers are gene products that are specifically expressed in the blood, urine, lymph, saliva, tumo...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N33/574
Inventor 周荣家程汉华陈恒玲
Owner WUHAN UNIV
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