Method for preparing RNA (ribonucleic acid) probe by using miR-155 precursor as template

A technology of RNA probe and RNA polymerase, which is applied in the field of preparing RNA probes to achieve the effect of reducing background signal, reducing background signal interference, and shortening experiment time

Inactive Publication Date: 2016-05-18
GUANGZHOU FULENGEN
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are deficiencies in the detection of the above-mentioned markers, so it is necessary to find more biomarkers. In recent years, the application of miRNA in the diagnosis of pancreatic cancer has become a hot spot of concern.

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  • Method for preparing RNA (ribonucleic acid) probe by using miR-155 precursor as template
  • Method for preparing RNA (ribonucleic acid) probe by using miR-155 precursor as template
  • Method for preparing RNA (ribonucleic acid) probe by using miR-155 precursor as template

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Embodiment 1

[0042] Example 1: Preparation of RNA probes using miR-155 precursor as a template

[0043] The process of preparing RNA probes using miR-155 precursor as a template can be found in figure 1 .

[0044] 1. Design of PCR primers:

[0045] (1) Find the name and sequence of its corresponding pre-miRNA in the miRBase database (http: / / www.mirbase.org / ), namely the miR-155 precursor (AccessionMI0000681): HomosapiensmiR-155stem-loop.

[0046] (2) Query the sequence of the corresponding pre-miRNA, and the result shows that the length of the pre-miRNA is about 100nt.

[0047] (3) Design a pair of primers.

[0048] Primer design:

[0049]

[0050] The primers mentioned in the above table are only examples, and those skilled in the art can design more suitable primers according to the principle of the present invention.

[0051] (4) Add the T7 promoter sequence to the 5' end of the reverse primer (see the table above) to ensure that the PCR product is complementary to the target fr...

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Abstract

The invention relates to a method for preparing an RNA (ribonucleic acid) probe by using an miR-155 precursor as a template. The method comprises the following steps: designing at least one pair of primers according to the sequence of the miR-155 precursor, wherein the PCR (polymerase chain reaction) amplification product of the primers is a multiplex DNA (deoxyribonucleic acid) segment, and the multiplex DNA (deoxyribonucleic acid) segment can comprise all target sequences; carrying out PCR amplification on the miR-155 precursor cDNA (complementary deoxyribonucleic acid) plasmid by adopting the primers in the step A to obtain the multiplex DNA segment, purifying, and mixing to obtain the template; adding an RNA polymerase and a biotin-labeled UTP (uridine triphosphate) to react, doping UTP into the product, and purifying to obtain the RNA probe. The cRNA (complementary ribonucleic acid) probe can be completely covered on all the target sequences. The probe preparation method simplifies the probe preparation steps, and greatly shortens the whole experimental time. The probe has the advantages of higher specificity, higher stability, weaker background signals and higher sensitivity. On such basis, the invention provides a kit for quickly detecting pancreatic-cancer-related miR-155 expression.

Description

technical field [0001] The invention belongs to the field of nucleic acid diagnosis, and in particular relates to a method for preparing an RNA probe using a miR-155 precursor as a template and an application thereof. Background technique [0002] Fluorescence in situ hybridization (FISH) is an important non-radioactive in situ hybridization technology. Fluorescent dye-labeled specific nucleic acid probes hybridize with corresponding target DNA or RNA molecules in cells. If the detected chromosomal target DNA and The nucleic acid probes used are homologous and complementary, and the two undergo denaturation-annealing-annealing. Since the DNA molecules on the chromosome are arranged linearly along the longitudinal axis of the chromosome, the probes can directly hybridize with the chromosome so that the specific Genes are located on chromosomes. By observing the fluorescent signal under a fluorescent microscope to determine the location of the DNA region or RNA molecule in th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/68
CPCC12Q1/6806C12Q1/6841C12Q2525/207C12Q2537/143C12Q2521/119C12Q2543/10
Inventor 徐学明鲁隼冯俊清
Owner GUANGZHOU FULENGEN
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