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Integrated microRNA fluorescent quantitative detection kit and application thereof

A kit and tiny technology, applied in the field of biological and medical research and clinical diagnosis, can solve the problems of non-specific amplification, primer specificity is not high enough, takes about 4-6 hours, etc.

Active Publication Date: 2016-11-23
金摩康(上海)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

However, this method has the following disadvantages: (1) Two-step reactions are required to complete cDNA synthesis. The first step is to poly(A) tail the microRNA, and the second step is to use Oligo(dT) primers to complete the reverse transcription reaction; (2) A detection process takes a long time, usually about 4-6 hours; (3) It is only used for the detection of miRNA, and has not been used for the detection of other microRNAs (including siRNA and piRNA); (4) The 3'-end universal primer is specific Not high enough, prone to non-specific amplification

Method used

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  • Integrated microRNA fluorescent quantitative detection kit and application thereof
  • Integrated microRNA fluorescent quantitative detection kit and application thereof
  • Integrated microRNA fluorescent quantitative detection kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0198] Determination of MicroRNA in Human Breast Cancer Cells

[0199] In this example, the total RNA from human breast cancer cell MDA-MB-231 (purchased from ATCC, USA) was prepared by conventional methods, and diluted to the required total RNA concentration by serial dilution method, 0.5 pg / 1 microliter , 5pg / 1 microliter, 50pg / 1 microliter, 500pg / 1 microliter. In the 20 microliter reaction system, the amount of total RNA is 0.5 pg, 5 pg, 50 pg or 500 pg.

[0200] Then use the method of the invention to quantitatively detect the microRNA content of the biological cell sample.

[0201] In this example, microRNA let-7b, let-7c and 5s rRNA (positive control) of human breast cancer cell line MDA-MB-231 were detected.

[0202] Amplification curve and melting curve when 5pg total RNA sample is used Figure 4 shown.

[0203] from Figure 4 A clear background signal stage, a fluorescent signal exponential amplification stage and an amplification plateau stage can be seen in the...

Embodiment 2

[0211] Determination of MicroRNA in Biological Fluid Samples

[0212] In this example, total RNA from human plasma (provided by healthy volunteers) was prepared by conventional methods, and the microRNA content of the biological fluid sample was quantitatively detected. Method is with embodiment 1. In the 20 microliter reaction system, the amount of total RNA is 5 pg or 50 pg.

[0213] For the amplification curve of the microRNA miR-16 (triple hole repeat, 50pg total RNA) of normal people's blood plasma such as Figure 5 shown.

[0214] from Figure 5 The result can be seen:

[0215] 1) The detection repeatability is good. The detection reproducibility of microRNA triplicate holes is very good.

[0216] 2) The detection accuracy is good. In this experiment, the sample RNA was diluted 1:10, and then the same microRNA miR-16 was detected simultaneously. Such as Figure 5 As shown, the difference between the amplification curves was 3.4 cycles. After converting to the ex...

Embodiment 3

[0220] Determination of MicroRNA in Biological Tissue Samples

[0221] In this example, conventional methods were used to prepare total RNA from the mammary fat pad tissue of mice (purchased from Shanghai Experimental Animal Center, Chinese Academy of Sciences), and quantitatively detect the microRNA content of the tissue samples. Method is with embodiment 1. In the 20 microliter reaction system, the amount of total RNA is 1 pg, 10 pg, or 50 pg.

[0222] The amplification curve and melting curve of the microRNA let-7b, let-7f and 5s rRNA (triple-well repeat, 10pg total RNA) of the mammary gland fat pad tissue of experimental mice are as follows Image 6 shown.

[0223] from Image 6 The results shown can be seen:

[0224] 1) The detection repeatability is good. The detection reproducibility of each microRNA triplicate well is very good.

[0225] 2) The detection specificity is good. Let-7b and let-7f belong to different members of the same tiny family, and their sequenc...

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Abstract

The invention provides an integrated microRNA fluorescent quantitative detection kit and application thereof. Particularly, the microRNA detection kit comprises a first reagent group for conducting a polyA tailing reaction and a reverse transcription reaction on microRNAs and a second reagent group for conducting PCR fluorescent quantitative detection, wherein the first reagent group comprises an enzyme for catalyzing the tailing reaction, an enzyme for catalyzing the reverse transcription reaction, a buffer solution for the tailing reaction and the reverse transcription reaction and a reverse transcription primer, and the second reagent group comprises a downstream universal primer which is used for a PCR amplification reaction and specifically combined to a universal sequence area of the reverse transcription primer. By adopting the kit, the microRNA tailing reaction and the reverse transcription reaction can be integrated into a whole. The kit can be used for quantitative detection on the miRNA, the siRNA and the piRNA and has a wide application prospect.

Description

technical field [0001] The invention relates to the fields of biological and medical research and clinical diagnosis, in particular to a microRNA fluorescence quantitative detection kit, and its application in the fluorescence quantitative detection of microRNA in tissue samples or blood samples, and the like. Background technique [0002] MicroRNA, including microRNA (miRNA), small interfering RNA (siRNA) and piwi-interacting RNA (piRNA), is a kind of non-coding single-stranded small molecule RNA with a length of about 18-30 nucleotides. [0003] miRNAs widely exist in eukaryotic organisms such as animals, plants, and nematodes. By specifically binding to the 3' non-coding region of the target gene mRNA, miRNA degrades the target mRNA or inhibits the translation of the target gene, resulting in a decrease in the expression of the target gene. miRNA participates in many physiological processes and pathological changes in the body, and regulates physiological processes such a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 王敏
Owner 金摩康(上海)生物技术有限公司