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A micro RNA expression detection method and its quantitative detection kit

A detection method and quantitative detection technology, applied in biochemical equipment and methods, microbial determination/inspection, fluorescence/phosphorescence, etc., can solve the problems of difficulty in the detection of MicroRNA molecule expression, easy degradation of MicroRNA, and complicated detection methods. Reduce primer-dimers and complex hairpin structures, the method is simple and efficient, and the effect of saving time and cost

Active Publication Date: 2016-03-09
上海尤里卡信息科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Therefore, the technical problem to be solved in the present invention is that it is relatively difficult to detect the expression of MicroRNA molecules. Detection method and a kind of MicroRNA quantitative detection kit

Method used

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  • A micro RNA expression detection method and its quantitative detection kit
  • A micro RNA expression detection method and its quantitative detection kit
  • A micro RNA expression detection method and its quantitative detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1 MicroRNA reverse transcription detection primers

[0070] The present invention provides 3 primers with a stem-loop structure for MicroRNA, and the names and sequences of the stem-loop primers are as follows:

[0071] Oligo1:

[0072] 5'-GTCGGAGAGACTGCAGTGGCGGACCAATTCGCACTCTCTCCGACTTTTTTTTA-3' (as shown in SEQ ID NO: 1 in the sequence listing);

[0073] Oligo2:

[0074] 5'-GTCGGAGAGACTGCAGTGGCGGACCAATTCGCACTCTCTCCGACTTTTTTTTC-3' (as shown in SEQ ID NO: 2 in the sequence listing);

[0075] Oligo3:

[0076] 5'-GTCGGAGAGACTGCAGTGGCGGACCAATTCGCACTCTCTCCGACTTTTTTTTTG-3' (as shown in SEQ ID NO: 3 in the sequence listing).

[0077] The above primers were prepared by Jierui Biosynthesis.

[0078] The present invention also utilizes MicroRNA universal reverse primer and Has-MiR-365a to detect forward primer, and described primer is as follows respectively:

[0079] MicroRNA universal reverse primer sequence: 5'-CTGCAGTGGCGGACCA-3' (as shown in SEQ ID NO: 4 in the...

Embodiment 2

[0085] Embodiment 2 experiment uses the preparation of each cell line

[0086] Sterile at 37°C, 5% CO 2 Under the same conditions, use 1640+10%FBS+1%SP medium for cell adhesion culture, culture Cal-27, Hep2, LOVO, MCF-7 cells respectively, in 6cm culture dishes, after the cells grow to about 80% , the microRNA extraction experiment can be carried out.

Embodiment 3

[0087] The preparation of embodiment 3MicroRNA sample

[0088] 1. Monolayer culture cells: absorb the culture medium, add BufferRLM (MicroRNA extraction buffer, Lot: 04911C; purchased from Kangwei Century, included in the MicroRNA extraction kit, Cat: CW0627), every 10cm 2 Add 1ml Buffer RLM.

[0089] 2. After adding BufferRLM to the sample, pipette it repeatedly several times to fully crack it. Leave at room temperature for 5 minutes to completely separate the protein-nucleic acid complex.

[0090] 3. Add chloroform at a ratio of 200 μl chloroform per 1ml BufferRLM, cover the tube cap, shake vigorously for 15 seconds, and place at room temperature for 5 minutes.

[0091] 4. Centrifuge at 12,000rpm at 4°C for 15 minutes. The sample is divided into three layers: a red organic phase, an intermediate layer and a colorless aqueous phase. Transfer the upper colorless aqueous phase to a new centrifuge tube.

[0092] 5. Add 1 / 3 times the volume of absolute ethanol to the solution ...

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Abstract

The invention discloses a detection method for expression of MicroRNA and a quantitative detection kit for MicroRNA. The method comprises the following steps: (1) extracting MicroRNA of a sample and connecting the MicroRNA to a Poly A tail; (2) mixing three primers with a stem-loop structure and then carrying out annealing treatment; (3) subjecting the MicroRNA connected with the Poly A tail and the primers with the stem-loop structure to annealing connection; (4) carrying out inverse transcription with the MicroRNA as a template so as to obtain cDNA; and (5) with the cDNA as a template, adding primers of an amplification target MicroRNA and detecting expression of the target MicroRNA by using a fluorescence quantitative PCR method. According to the detection method, all the MicroRNA and reference genes of a to-be-detected object can be obtained through one inverse transcription, so generation of a primer dimer and a complex hairpin structure is substantially reduced, and detection efficiency of MicroRNA is greatly improved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a MicroRNA expression detection method and a quantitative detection kit thereof. Background technique [0002] MicroRNA is a type of non-coding single-stranded RNA found in a variety of eukaryotic cells and viruses in recent years. It is a short sequence of 21-25 nt in length and is highly conserved in evolution. It can pass Complementary pairing with target mRNA-specific bases, causing target mRNA degradation or inhibiting its translation, thereby regulating gene expression after transcription (O`DonnellKA, WentzelEA, ZellerKI, etal.e-Myc-regulatedmicroRNAsmodulateE2F1expression[J].Nature , 2005,4(7043):839.). Due to the certain degree of conservation of MicroRNA sequences in different organisms, it is generally believed that the function of MicroRNA is to participate in some basic processes of life, such as cell proliferation, cell death, stress response and fat metabo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68G01N21/64
Inventor 姚远颋王海峰费倩岚谈竹君劳昕元
Owner 上海尤里卡信息科技有限公司