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Colorimetric method for simultaneously detecting multiple miRNA (micro-ribonucleic acid) sequences based on competitive hybridization reaction

A technology of hybridization reaction and DNA sequence, which is applied in biochemical equipment and methods, measuring devices, microbe determination/inspection, etc., and can solve problems such as low sensitivity, inability to distinguish homologous miRNA sequences, and time-consuming

Inactive Publication Date: 2014-01-22
CENT SOUTH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, Northern blotting is a semi-quantitative detection method with cumbersome steps, time-consuming and low sensitivity
PCR technology can perform highly sensitive and specific quantitative detection of miRNA, but the method is cumbersome and requires pretreatment of miRNA
Although the Microarray method can quickly and high-throughput detect a large number of miRNA sequences in a short period of time, it is expensive and cannot distinguish homologous miRNA sequences with small base differences.

Method used

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  • Colorimetric method for simultaneously detecting multiple miRNA (micro-ribonucleic acid) sequences based on competitive hybridization reaction
  • Colorimetric method for simultaneously detecting multiple miRNA (micro-ribonucleic acid) sequences based on competitive hybridization reaction
  • Colorimetric method for simultaneously detecting multiple miRNA (micro-ribonucleic acid) sequences based on competitive hybridization reaction

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Effect of hybridization temperature (determination of miRNA-182 sequence):

[0059] (1) Add 60 μL of 1 nM amino DNA solution (sequence 3'-AAA CCG TTA CCA TCT TGA GTG TGA TTT TT-(CH 2 ) 6 NH 2 -5'), let stand overnight, and wash with PBS buffer several times;

[0060] (2) Block with 180 μL of 2% BSA solution at room temperature for 2.5 hours, and then wash with PBS buffer several times;

[0061] (3) On the basis of the above step (2), add 120 μL of biotin-labeled miRNA-182 solution (sequence 3'-UCA CAC UCA AGA UGG UAA CGG UUU-biotin-5') with a concentration of 1 nM dropwise, respectively Placed at 25°C, 30°C, 37°C, 45°C, 50°C, reacted for 4 hours, washed with PBS buffer several times;

[0062] (4) On the basis of the above step (3), add 60 μL SA-Poly-HRP solution dropwise, and after standing at room temperature for 1 hour, wash with PBS buffer several times;

[0063] (5) Mix 100 μL of 0.1% TMB solution and 25 μL of 0.03% H 2 o 2 The solution was successively added...

Embodiment 2

[0080] The influence of hybridization temperature and hybridization time is as embodiment 1;

[0081] The content detection experiment of miRNA-185 (sequence 5'-UGG AGA GAA AGG CAG UUC CUGA-3') in serum:

[0082] Effect of different concentrations of biotin-labeled miRNA sequence (identical to miRNA-185 sequence):

[0083] (1) Add 60 μL of 1 nM amino DNA solution dropwise to the microtiter plate, let it stand overnight, and wash it with PBS buffer several times;

[0084] (2) Block with 180 μL of 2% BSA solution at room temperature for 2.5 hours, and then wash with PBS buffer several times;

[0085] (3) On the basis of step (2), drop a series of 120 μL biotin-miRNA solutions with different concentrations in the microwells, the concentrations are 0, 0.1, 0.2, 0.5, 0.8, 1.0, 2.0, 5.0, 10 nM , placed at 37°C for 2.5 hours, washed with PBS buffer several times;

[0086] (4) On the basis of step (3), add 60 μL of SA-Poly-HRP solution dropwise into the microwells, let stand at roo...

Embodiment 3

[0092] The influence of hybridization temperature and hybridization time is as embodiment 1;

[0093] The content detection experiment of miRNA-381 (sequence is 5'-UAU ACA AGG GCA AGC UCU CUGU-3') in serum:

[0094] The effect of different concentrations of biotin-labeled miRNA sequence (same sequence as miRNA-381):

[0095] (1) Add 60 μL of 10 nM amino DNA solution dropwise to the microtiter plate, let it stand overnight, and wash it with PBS buffer several times;

[0096] (2) Block with 180 μL of 2% BSA solution at room temperature for 2.5 hours, and then wash with PBS buffer several times;

[0097] (3) On the basis of step (2), drop a series of 120 μL biotin-miRNA solutions with different concentrations in the microwells, the concentrations are 0, 0.1, 0.2, 0.5, 0.8, 1.0, 2.0, 5.0, 10 nM , placed at 37°C for 2.5 hours, then rinsed with PBS buffer several times;

[0098] (4) On the basis of step (3), add 60 μL of SA-Poly-HRP solution dropwise into the microwells, let stan...

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Abstract

The invention discloses a colorimetric method for simultaneously detecting multiple miRNA (micro-ribonucleic acid) sequences based on a competitive hybridization reaction. The method comprises the following steps: assembling amino modified DNA (deoxyribonucleic acid) on the surface of a micropore plate, and closing micropores; performing a competitive hybridization reaction on biotin modified miRNA and a target miRNA sequence with a fixed DNA probe on the surface of the micropores; deriving avidin marked poly-horseradish peroxidase to the surface of the micropores, and catalyzing a chromogenic reaction between TMB (tetramethylbenzidine) and hydrogen peroxide by the poly-horseradish peroxidase. The target miRNA sequence is detected by determining the absorbance of a colored solution. The method is simple, quick, high in sensitivity and good in selectivity, does not need to mark an actual sample, and can be directly used for detecting multiple miRNA sequences.

Description

technical field [0001] The invention relates to a colorimetric method for simultaneously detecting multiple miRNA sequences based on competitive hybridization reactions, and belongs to the technical field of miRNA sequence detection. Background technique [0002] MicroRNA (miRNA) is a group of short endogenous small RNA molecules that do not encode proteins, and its length is about 17-25 nucleotides (nt). The production of miRNA in living organisms is a complex process. First, pri-miRNA (primary miRNA) forms pre-miRNA (precursor miRNA) with a hairpin structure under the action of Drosha enzyme in the nucleus; then, pre-miRNA is transformed into Mature miRNA containing 17-25 nucleotides. More than 1000 miRNA sequences have been detected, but most miRNAs have similar sequences and functions. [0003] At present, the detection of miRNA is mainly based on hybridization and amplification methods. Qualitative and quantitative detection techniques mainly include: Northern blott...

Claims

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Application Information

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IPC IPC(8): G01N33/558G01N21/31
CPCC12Q1/6837C12Q2525/207C12Q2565/501
Inventor 王建秀张宇冯城婷
Owner CENT SOUTH UNIV
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