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Preparation method of kit for joint detection of esophagus cancer by using four novel circRNAs (ribonucleic acids)

A joint detection and esophageal cancer technology, applied in DNA/RNA fragments, biochemical equipment and methods, recombinant DNA technology, etc., can solve the problem that the survival rate is less than 20%

Active Publication Date: 2020-06-19
王强
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although my country has attached great importance to the diagnosis and treatment of esophageal cancer for many years, 90% of the diagnosed EC patients are in the middle and late stage, and the 5-year survival rate is less than 20%. esophageal cancer related
However, due to the limitation of detection technology, circRNA was considered to be the experimental interference product formed by the missplicing of exon transcripts at that time, which did not attract people's attention

Method used

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  • Preparation method of kit for joint detection of esophagus cancer by using four novel circRNAs (ribonucleic acids)
  • Preparation method of kit for joint detection of esophagus cancer by using four novel circRNAs (ribonucleic acids)
  • Preparation method of kit for joint detection of esophagus cancer by using four novel circRNAs (ribonucleic acids)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] RNA extraction:

[0064] The blood and tissue samples to be tested come from cancer patients and normal people, and the cancer is esophageal cancer. The RNAprep Pure Hi-Blood (tissue) Kit RNAprep Pure kit from TIANGEN Company was used for RNA extraction from blood (tissue).

[0065] The RNA extraction steps in the blood and tissue samples to be tested are as follows:

[0066] (1) Blood RNA extraction:

[0067] (1) Dilution of erythrocyte lysate: select an appropriate volume of 10× erythrocyte lysate H according to the volume of the processed blood sample (for example, if the volume of the blood sample to be processed is 200 μL, take 140 μL 10× erythrocyte lysate H), and use RNase- Free ddH2O diluted to 1 × red blood cell lysate H;

[0068] (2) Add 5 times the volume of 1× red blood cell lysate H to (1) volume of human whole blood (you need to prepare a suitable clean tube);

[0069] (3) Incubate on ice for 10-15 minutes, and vortex and shake twice during the incubat...

Embodiment 2

[0098] Reverse transcription reaction system

[0099] The reverse transcription reaction system is as follows:

[0100] Reverse transcription reaction system components Usage amount RT mix 4.0 μL total RNA 1μg RNase-free ddH2O Add ddH2O to a total reaction volume of 16 μL

Embodiment 3

[0102] qPCR operation reaction system

[0103] The qPCR operation reaction system is as follows:

[0104] Reaction system components Usage amount mix 5.0 μL F 0.5μL R 0.5μL H2O 2.0 μL RNase-free ddH2O 2.0 μL total capacity 10.0 μL

[0105] Reverse transcription reaction conditions were: 37°C for 15min, 85°C for 5s, 4°C∞; fluorescent quantitative PCR reaction conditions were stage 1: 50°C for 2min, 1 cycle; stage 2: 95°C for 10min, 1 cycle; stage 3: 95°C 15s at ℃, 1min at 60℃, 40 cycles; Stage 4: 15s at 95℃, 1min at 60℃, 15s at 95℃, 15s at 60℃, 1 cycle.

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Abstract

The invention discloses a preparation method of a kit for joint detection of esophagus cancer by using four novel circRNAs (ribonucleic acids). The sequences of the four novel circRNAs are shown as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 respectively. The expression conditions of the four genes such as novel circRNA_1, 2, 3 and 4 in blood of esophagus cancer patients and normal people are verified in tissues, and the detection of the novel circRNA_1, 2, 3 and 4 genes is realized through a fluorescent quantitative PCR optimal system; and results show that the joint detection of the novel circRNA_1, 2, 3 and 4 has diagnosis significance, and can be used for esophagus cancer diagnosis, treatment effect evaluation or prognosis condition evaluation.

Description

technical field [0001] The invention belongs to the technical field of molecular diagnosis, and in particular relates to a preparation method of a kit for jointly detecting esophageal cancer by four novel circRNAs. Background technique [0002] Esophageal cancer (EC) is one of the most common malignant tumors of the digestive system, and refers to malignant tumors that originate in the esophageal epithelium. my country is a country with a high incidence of esophageal cancer, ranking among the top five in the world, much higher than the average level in Asia and the world. Although my country has attached great importance to the diagnosis and treatment of esophageal cancer for many years, 90% of the diagnosed EC patients are in the middle and advanced stages, and the 5-year survival rate is less than 20%. associated with esophageal cancer. Therefore, an in-depth understanding of the molecular mechanism of tumor development and supplementation and improvement of the existing ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/6851C12N15/113
CPCC12Q1/6886C12Q1/6851C12Q2600/178C12Q2600/118C12Q2600/158C12Q2531/113C12Q2563/107
Inventor 王强耿冲徐淑芠李龙飞
Owner 王强