Double-recognition quantitative detection method for microRNA

A quantitative detection method and quantitative detection technology, applied in the fields of biotechnology and clinical medical diagnosis, can solve the problems of labor consumption, time-consuming detection of miRNA, technical difficulty in large-scale expression analysis, etc., to avoid background fluorescence and poor detection performance Falling, good stability effect

Inactive Publication Date: 2017-03-22
NANJING UNIVERSITY OF TRADITIONAL CHINESE MEDICINE
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the difference in melting point temperature of miRNAs caused by sequence diversity makes hybridization-based techniques difficult to apply to large-scale expression analysis, and PCR-based techniques require multiple procedures such as miRNA labeling and complementary DNA conversion steps, making Detecting miRNA is time-consuming and labor-intensive

Method used

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  • Double-recognition quantitative detection method for microRNA
  • Double-recognition quantitative detection method for microRNA
  • Double-recognition quantitative detection method for microRNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: Upconverting Nanoparticles (NaYF 4 : Er 3+ , Yb 3+ ) labeled oligonucleotide (LRET-B)

[0024]Experimental materials: bovine serum albumin, batch number 140318, Shanghai Aladdin Reagent Co., Ltd.; 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), batch number 090M14531V, Shanghai Aladdin Reagent Co., Ltd. Latin Reagent Co., Ltd.; Succinimide (NHS), batch number MKBG7914V, Shanghai Aladdin Reagent Co., Ltd.; Ethanol, batch number: 14021710247, purchased from Nanjing Chemical Reagent Co., Ltd. The oligonucleotide sequence (eg 5'-TTGTGTTCCGTAGGCT-AAAAAAAA 3'C7-NH2) from the 5' end is the specific sequence that is complementary to the recognition unit, the sequence that controls the length of the oligonucleotide, and the 3' end modified C7 amino The sequence composition, in which TTGTGTTCCGATAGGCT is a specific sequence that is complementary to the recognition unit, varies with the detected miRNA. The oligonucleotide sequence is:

[0025] UC-L...

Embodiment 2

[0041] Example 2: Quantitative detection of miRNA-21 based on dual recognition microRNAs

[0042] Experimental material: ethanol, batch number: 14021710247, purchased from Nanjing Chemical Reagent Co., Ltd. HEPES buffer, batch number: E607018, purchased from Shanghai Sangon Biological Reagent Co., Ltd.; 5′ adenosine triphosphate disodium salt trihydrate (ATP), batch number: A600020, purchased from Shanghai Sangon Biological Reagent Co., Ltd.; single stranded salmon sperm sperm DNA, batch number: C640031, was purchased from Shanghai Sangon Biological Reagent Co., Ltd.; RNA ligase, batch number: B101801, was purchased from Shanghai Sangon Biological Reagent Co., Ltd.

[0043] The oligonucleotide sequence is:

[0044] UC-LRET-B: 5’-TTGTGTTCCGATAGGCT-AAAAAAAA 3’ C7-NH 2 ;

[0045] UC-LRET-A: Cy3-5’ AAAAAAAA-CGATCAGTCAGGCAA-3’ C6;

[0046] the adaptor oligos (for miRNA-21):

[0047] (RD-REG-A): 3’ TCCCCAACACAAGGCTATCCGA-ATCGAATAGTCT-5’-PO 4

[0048] (RD-REG-D): 3’ OH-GACTACA...

Embodiment 3

[0052] Example 3: Quantitative detection of miRNA-125a based on dual recognition microRNAs

[0053] Experimental material: ethanol, batch number: 14021710247, purchased from Nanjing Chemical Reagent Co., Ltd. HEPES buffer, batch number: E607018, purchased from Shanghai Sangon Biological Reagent Co., Ltd.; 5′-adenosine triphosphate disodium salt trihydrate (ATP), batch number: A600020, purchased from Shanghai Sangon Biological Reagent Co., Ltd.; single stranded salmon sperm DNA, batch number: C640031, purchased from Shanghai Sangon Biological Reagent Co., Ltd.; RNA ligase, batch number: B101801, purchased from Shanghai Sangon Biological Reagent Co., Ltd.;

[0054] The oligonucleotide sequence is:

[0055] UC-LRET-B: 5’-TTGTGTTCCGATAGGCT-AAAAAAAA 3’ C7-NH 2 ;

[0056] UC-LRET-A: Cy3-5’ AAAAAAAA-CGATCAGTCAGGCAA-3’ C6;

[0057] the adaptor oligos (for miRNA-125a):

[0058] (RD-REG-A): 3’ TCCCCAACACAAGGCTATCCGA-AGGGACTCTGGG-5’-PO 4

[0059] (RD-REG-D): 3' OH-AAATTGGACACT-GCT...

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Abstract

The invention discloses a method for achieving micronRNA single-step, phase homogenizing and high-sensitivity detection by combining different double recognition processes of hybridization and ligation based on an upconversion luminescence resonance energy transfer (UC-LRET) technology. The method is mainly applied to microRNA expression profiles analysis, microRNA clinical diagnosis, microRNA study detection and microRNA related pharmaceutical studying and screening. The method has the following beneficial effects that the number of operation steps is small, and the method belongs to one-step method detection; the stability is good, long-time storage can be achieved, and the detecting performance is not reduced; and stimulating occurs in a near infrared region (980 nm), so that generation of background fluorescence is avoided. Thus, the method has very good application prospects.

Description

technical field [0001] The invention belongs to the fields of biotechnology and clinical medical diagnosis, and relates to a quantitative detection method for double-identification detection of microRNA. Based on upconversion fluorescence resonance energy transfer (UC-LRET) technology combined with different hybridization and ligation dual recognition detection, it can achieve single-step, homogeneous and highly sensitive detection of microRNA. Mainly used in microRNA expression profiling, microRNA clinical diagnosis, microRNA research and detection, and microRNA-related drug research and screening. Background technique [0002] MicroRNA (hereinafter referred to as "miRNA") is a class of non-coding RNAs of approximately 20-24 nucleotides (nt) in length, which can regulate more than half of human genes. [See (a) D.P.Bartel, Cell, 2009, 136, 215-233. (b) S.L.Ameres, M.D.Horwich, J.H.Hung, J.Xu, M.Ghildiyal, Z.Weng and P.D.Zamore, Science, 2010, 328 , 1534-1539.(c)Y.Chen, D.Y...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6809C12Q2563/107C12Q2563/149
Inventor 朱栋胡玥张小静
Owner NANJING UNIVERSITY OF TRADITIONAL CHINESE MEDICINE
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