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Hsa-miR-629-5p detection kit based on AllGlo probe fluorescent quantitative PCR (Polymerase Chain Reaction) and detection method thereof

A cel-mir-39 and detection kit technology, applied in the field of MicroRNA, can solve the problems of expensive synthesis and unfavorable promotion, and achieve rapid and accurate detection, low background signal, high specificity and sensitivity

Active Publication Date: 2014-04-30
ANHUI IPROCOM BIOTECH CO LTD
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  • 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

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  • Hsa-miR-629-5p detection kit based on AllGlo probe fluorescent quantitative PCR (Polymerase Chain Reaction) and detection method thereof
  • Hsa-miR-629-5p detection kit based on AllGlo probe fluorescent quantitative PCR (Polymerase Chain Reaction) and detection method thereof
  • Hsa-miR-629-5p detection kit based on AllGlo probe fluorescent quantitative PCR (Polymerase Chain Reaction) and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] See figure 1 The embodiment of the hsa-miR-629-5p detection kit based on AllGlo probe fluorescence quantitative PCR of the present invention is provided with a box body 1, a partition 2, an exogenous reference bottle 3, and a stem-loop reverse transcription reagent bottle 4 , Real-time fluorescent quantitative PCR reagent bottle 5; the partition 2 is set in the box 1, the exogenous reference bottle 3, the stem-loop reverse transcription reagent bottle 4, and the real-time fluorescent quantitative PCR reagent bottle 5 are inserted on the partition 2. The sex reference bottle 3 contains an exogenous reference, the stem-loop reverse transcription reagent bottle 4 contains a stem-loop reverse transcription reagent, and the real-time fluorescent quantitative PCR reagent bottle 5 contains a real-time fluorescent quantitative PCR reagent.

[0052] ① The exogenous reference can be cel-miR-39, etc., the cel-miR-39 is a nematode miRNA, and its working concentration can be 5n mol, an...

Embodiment 2

[0056] The detection of serum or plasma hsa-miR-629-5p includes the following steps:

[0057] 1. Collect plasma or serum:

[0058] Take 2 mL of fresh blood sample and put it in EDTA anticoagulant tube or tube without anticoagulant. Immediately turn the above test tube upside down and mix for 6-8 times, then place the above test tube in a centrifuge at 3000r for 10 minutes, then take it out and place it in a test tube rack. Put the supernatant into a new 1.5mL RNase-free centrifuge tube. Place the 1.5mL centrifuge tube containing the supernatant in a centrifuge at 13000r for 10 minutes. After the end, 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 in this step.), aliquot 400-500μL from each tube, take 200μL for the next extraction, and freeze the remaining plasma or serum at -80℃ Save.

[0059] 2. Extract microRNA

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

Embodiment 3

[0082] Example 3. Tissue or cell miRNA detection

[0083] 1. Sample processing:

[0084] a. Tissue: Grind the tissue in liquid nitrogen. Add 1 mL of Lysis Solution MZ (manufactured by Tiangen Biotechnology Co., Ltd.) for every 30-50mg animal tissue or 100mg plant tissue, and homogenize with a homogenizer. The sample volume should not exceed 10% of the MZ volume of the lysis buffer.

[0085] b. Monolayer cultured cells: directly add lysis buffer MZ to the culture plate to lyse the cells, every 10cm 2 Add 1mLMZ to the area. Use the sampler to beat several times.

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

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

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

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Abstract

A hsa-miR-629-5pdetection kit based on AllGlo probe fluorescent quantitative PCR (Polymerase Chain Reaction) and a detection method of the hsa-miR-629-5pdetection kit relate to MicroRNA. The detection kit is provided with a kit body, a clapboard, an exogenous reference bottle, a stem-loop reverse transcription reagent bottle, and a real-time fluorescent quantitative PCR reagent bottle. The miRNA in a sample is extracted, and if the sample is a serum / plasma sample or other liquid samples, then 5 mu L exogenous reference cel-miR-39 with the concentration of 5nmol, provided by the reagent bottle, is added after the sample is fully split, and the mixture is oscillated in vortex; but if the sample is a cell or tissue sample, then exogenous reference cel-miR-39 is not added; a stem-loop reverse transcription reagent provided by the reagent bottle is used to reversely transcribemiRNA into cDNA; a real-time fluorescent quantitative PCR reagent provided by the reagent bottle is used to conduct real-time PCR amplification; various data provided by an analytical instrument are integrated together, and reasonable threshold and reference line are set for result analysis.

Description

Technical field [0001] The invention relates to MicroRNA, in particular to a hsa-miR-629-5p detection kit based on AllGlo probe fluorescence quantitative PCR and a detection method thereof. Background technique [0002] MicroRNA (miRNA) is a type of non-coding RNA molecule with a length of about 22 nucleotides that is widely present in eukaryotic cells. It participates in many physiological and pathological processes in organisms. By regulating gene expression, it can promote cell proliferation and apoptosis. , Growth and development, cell differentiation, metabolism, etc., play an important role in the process of miRNA in the nucleus after forming the initial primary transcript pri-miRNA to pre-miRNA, and then transported out of the nucleus, through shearing to form mature miRNA, through Forms RISC (RNA-induced silencing complex) with Ago protein, etc., partially inhibits or degrades target mRNA sequences, and participates in gene expression regulation (Bartel DP.MicroRNAs:genom...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12M1/34
CPCC12Q1/686C12Q2561/113C12Q2563/107
Inventor 牛建军唐晶张忠英
Owner ANHUI IPROCOM BIOTECH CO LTD
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