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Serum miR-21 (micro-ribonucleic acid-21) detection kit based on luorescence quantitative PCR (polymerase chain reaction) and application thereof

A fluorescent quantitative detection, mir-21 technology, applied in fluorescence/phosphorescence, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problems of reducing detection accuracy, reducing cost, increasing reaction non-specificity, etc. Low, easy to operate, stable performance

Inactive Publication Date: 2012-02-08
BEIJING CANCER HOSPITAL PEKING UNIV CANCER HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In real-time fluorescent quantitative PCR, the SYBR Green I dye method can greatly reduce the cost compared with the Taqman probe method used by ABI, but at the same time it inevitably increases the non-specificity of the reaction and reduces the accuracy of detection.

Method used

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  • Serum miR-21 (micro-ribonucleic acid-21) detection kit based on luorescence quantitative PCR (polymerase chain reaction) and application thereof
  • Serum miR-21 (micro-ribonucleic acid-21) detection kit based on luorescence quantitative PCR (polymerase chain reaction) and application thereof
  • Serum miR-21 (micro-ribonucleic acid-21) detection kit based on luorescence quantitative PCR (polymerase chain reaction) and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Embodiment 1Trizol reagent is prepared

[0060] Trizol reagent preparation, first mix 100g of guanidine isothiol, 118mL of distilled water, 7.04mL of 0.75M sodium citrate, 10.56mL of 10% sodium lauryl sarcosinate, and 20mL of 2M sodium acetate, stir and dissolve; Put 200mL of redistilled phenol (crystal at room temperature) in a water bath at 60°C until completely melted, then add 0.2g of 8-hydroxyquinoline and 1.2g of β-mercaptoethanol or 0.5g of dithiothreitol; then mix all the above reagents , stirred thoroughly, and finally adjusted the pH value to 4.6, and stored at 4°C. The shelf life is about 9 months.

Embodiment 2

[0061] The extraction of embodiment 2 serum RNA

[0062] (1) Take 33 cases of breast cancer patients and 49 cases of healthy people and put them into 2mL EP tubes that have been treated with DEPC water, then add 1mL Trizol reagent prepared in Example 1, and 200 μL chloroform, vortex fully Homogenize for 1 min (note that if the vortex time is not enough or the strength is not strong, the layers will not be well layered). Then let stand for 3min.

[0063] (2) Centrifuge the EP tube at 12000 rpm at 4°C for 15 minutes. Carefully pipette 600 μL of the upper aqueous phase into a 1.5mL EP tube (do not touch the white layer in the middle, otherwise it will easily lead to protein contamination). Then add 600 μL of pre-cooled isopropanol, mix thoroughly, and centrifuge at 4°C for 15 minutes.

[0064] (3) Pour off the centrifuged liquid carefully. The remaining liquid can be centrifuged for a while and then sucked up with a 200mL gun, but try not to touch the tube wall and bottom. Ad...

Embodiment 3

[0066] Example 3 reverse transcription of miR-21 and miR-16

[0067] The RNA extracted in Example 2 was reverse-transcribed with a self-assembled reverse transcription kit. Add 23 μL of extracted RNA and 23 μL of DEPC water [as a reverse transcription no-template control (RT-NTC)] to various reagents in the reverse transcription kit, which contain a specific volume of 500 nmol / L miR-21 or miR-16 stem-loop Reverse transcription primer 1μL, 200U / μL M-MLV 1μL, reverse transcription buffer 2μL, 1mol / 1DTT 0.1μL, 10mmol / 1DNTPs 0.5μL and 40U / μL RNAsin 0.1μL. The reaction conditions were: annealing at 16°C for 30 minutes, reverse transcription at 42°C for 60 minutes, and reverse enzyme inactivation at 85°C for 5 minutes. Then, the reverse transcription product was stored at -20°C.

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Abstract

The invention provides a real-time serum miR-21 (micro-ribonucleic acid-21) detection kit based on fluorescence quantitative PCR (polymerase chain reaction). The kit comprises a reverse transcription primer shown in SEQ ID No.1, PCR primers shown in SEQ ID No.2 and 3, a reverse transcription primer shown in SEQ ID No.4 and PCR primers shown in SEQ ID No.5 and 6, as well as a Trizol reagent, a reverse transcription reagent and a PCR amplification reagent. The invention also provides a method applying the serum miR-21 detection kit. The method for detecting miR-21 in serum of mammary cancer hasthe advantages of convenience in operation, low cost, sensitive diagnosis and quantitative detection, can be used for conveniently detecting the miR-21 in the serum, and is suitable for auxiliary diagnosis and relapse detection of a patient with mammary cancer.

Description

technical field [0001] The invention belongs to the field of biological detection, and in particular relates to a serum miR-21 detection kit and its application in auxiliary diagnosis of breast cancer. Background technique [0002] miRNA is a kind of endogenous non-coding small molecule single-stranded RNA with a length of 19-24 nucleotides (nt), which is highly conserved in the evolution process and can cause specific base complementary pairing with target gene mRNA. The degradation of mRNA or inhibition of its translation generally negatively regulates the expression of target genes. A large number of experiments have proved that miRNA plays an important role in life activities such as stem cell maintenance, cell differentiation, proliferation, apoptosis, metabolism, embryonic development, immune response and tumor development. In recent years, miRNA has been regarded as a new generation of tumor markers, which play a very important role in tumor diagnosis and prognosis. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11G01N21/64
Inventor 张青云李雪峰
Owner BEIJING CANCER HOSPITAL PEKING UNIV CANCER HOSPITAL
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