Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Hsa-mir-9 detection kit and detection method based on allglo probe fluorescent quantitative PCR

A technology of hsa-mir-9 and cel-mir-39, which is applied in the field of hsa-miR-9 detection kits, can solve the problems of unfavorable promotion, high synthesis price, etc., and achieves low background signal, shortened reaction time, and synthesis. Procedurally simple effects

Inactive Publication Date: 2016-03-23
ZHONGSHAN HOSPITAL XIAMEN UNIV
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The reverse transcription primer based on the stem-loop structure can specifically recognize and amplify the mature miRNA sequence, but the precursor of the miRNA has not been amplified. The disadvantage of Taqman-MGB probe detection of miRNA is that the synthesis is expensive, which is not conducive to popularization.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Hsa-mir-9 detection kit and detection method based on allglo probe fluorescent quantitative PCR
  • Hsa-mir-9 detection kit and detection method based on allglo probe fluorescent quantitative PCR
  • Hsa-mir-9 detection kit and detection method based on allglo probe fluorescent quantitative PCR

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] see figure 1 , the embodiment of the hsa-miR-9 detection kit based on AllGlo probe fluorescent quantitative PCR of the present invention is provided with a box body 1, a partition 2, an exogenous reference bottle 3, a stem-loop reverse transcription reagent bottle 4, and a real-time Fluorescent quantitative PCR reagent bottle 5; partition 2 is set in the box body 1, exogenous reference bottle 3, stem-loop reverse transcription reagent bottle 4, real-time fluorescent quantitative PCR reagent bottle 5 are inserted on the partition 2, exogenous reference Bottle 3 is equipped with exogenous reference, stem-loop reverse transcription reagent bottle 4 is equipped with stem-loop reverse transcription reagent, and real-time fluorescent quantitative PCR reagent bottle 5 is equipped with real-time fluorescent quantitative PCR reagent.

[0052] ① The exogenous reference can be cel-miR-39, etc., the cel-miR-39 is a kind of nematode miRNA, its working concentration can be 5nmol, its...

Embodiment 2

[0056] The detection of serum or plasma hsa-miR-9 comprises the following steps:

[0057] 1. Collect plasma or serum:

[0058] Take 2mL of fresh blood samples and put them in EDTA anticoagulant tubes or anticoagulant-free tubes. Immediately invert the above test tubes and mix them 6-8 times, then place the above test tubes in a centrifuge at 3000rr for 10min, take them out and place them in a test tube rack First, carefully draw the supernatant and put it into a new 1.5mL RNase-free centrifuge tube, place the 1.5mL centrifuge tube containing the supernatant in a centrifuge at 13000r for 10min, and transfer the upper plasma or serum to In a new 1.5mL RNase-free centrifuge tube (be careful not to absorb the cell pellet in the lower layer during this step), aliquot 400-500μL for each tube, take 200μL for the next step of extraction, and freeze the remaining plasma or serum at -80°C live.

[0059] 2. Extract microRNA

[0060] Use the miRNA extraction kit (DP501) produced by Tia...

Embodiment 3

[0082] Example 3. Tissue or cell miRNA detection

[0083] 1. Sample handling:

[0084] a. Tissue: Triturate the tissue in liquid nitrogen. For every 30-50 mg of animal tissue or 100 mg of plant tissue, add 1 mL of lysate MZ (manufactured by Tiangen Biotechnology Co., Ltd.), and use a homogenizer for homogenization. The sample volume should not exceed 10% of the MZ volume of the lysate.

[0085] b. Monolayer culture cells: directly add lysate MZ to the culture plate to lyse the cells, every 10cm 2 Add 1mL LMZ to the area. Swipe several times with a sampler.

[0086] c. Cell suspension: centrifuge for 800r5min to take the cells, and discard the supernatant. Add 1mL Lysis Buffer MZ, oscillate or pipette several times to mix. Do not wash the cells before adding Lysis Buffer MZ to avoid RNA degradation.

[0087] 2. Tissue, cell miRNA extraction and enrichment

[0088] The miRNA extraction kit (DP501) produced by Tiangen Biotechnology Co., Ltd. was used to extract tissue or ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses an hsa-miR-9 (Human Serum Albumin-Micro Ribonucleic Acid-9) detection kit and an hsa-miR-9 detection method based on AllGlo probe fluorescent quantitative PCR (Polymerase Chain Reaction) and relates to MicroRNA (Micro Ribonucleic Acid). The kit is provided with a kit body, a partition, an exogenous reference bottle, a stem-loop reverse transcription reagent bottle and a real-time fluorescent quantitative PCR reagent bottle. The detection method comprises the steps of extracting miRNA from a sample, adding 5 microliters of exogenous reference cel-miR-39 (Caenorhabditis Elegans-Micro Ribonucleic Acid-39) at a concentration of 5n mol provided by the kit after full lysis of the sample, and swirling and shaking in case of a sample of serum / plasma sample or other body fluids, adding no exogenous reference cel-miR-39 in case of a sample of a cell or a tissue, performing reverse transcription on miRNA to form cDNA (Complementary Deoxyribonucleic Acid) with a stem-loop reverse transcription reagent provided by the kit, performing real-time PCR amplification on cDNA with a real-time fluorescent quantitative PCR reagent provided by the kit, and setting a reasonable threshold and a baseline for result analysis by synthesizing various data given by an analytical instrument.

Description

technical field [0001] The invention relates to MicroRNA, in particular to a hsa-miR-9 detection kit and detection method based on AllGlo probe fluorescence quantitative PCR. Background technique [0002] MicroRNA (miRNA) is a kind of non-coding RNA molecule of about 22 nucleotides widely present in eukaryotic cells, which participates in many physiological and pathological processes in organisms, by regulating gene expression, in cell proliferation, apoptosis , growth and development, cell differentiation, metabolism and other processes play an important role. Specifically, miRNA forms the initial primary transcript pri-miRNA to pre-miRNA in the nucleus, and then transports out of the nucleus to form mature miRNA through cleavage. Form RISC (RNA-induced silencing complex) with Ago protein, partially inhibit or degrade target mRNA sequence, and participate in gene expression regulation (BartelDP. MicroRNAs: genomics, biogenesis, mechanism, and function [J]. Cell, 2004, 116 (...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12M1/34
CPCC12Q1/686C12Q2561/113C12Q2563/107
Inventor 唐晶刘文娟张忠英
Owner ZHONGSHAN HOSPITAL XIAMEN UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com