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DNA molecular probe and application on miRNA real-time quantification PCR detection of cancer cell of S1 nuclease

A technology of DNA molecules and molecular probes, applied in the field of miRNA detection in tumor cells, can solve the problems of increasing experimental errors, many experimental steps, time-consuming detection errors, etc.

Inactive Publication Date: 2018-05-18
CHINA THREE GORGES UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method still needs reverse transcription reaction to amplify the first cDNA strand of miRNA, so there are many experimental steps, which will inevitably increase experimental errors; when this method is used in high-throughput screening experiments, it is time-consuming and has detection errors. bigger

Method used

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  • DNA molecular probe and application on miRNA real-time quantification PCR detection of cancer cell of S1 nuclease
  • DNA molecular probe and application on miRNA real-time quantification PCR detection of cancer cell of S1 nuclease
  • DNA molecular probe and application on miRNA real-time quantification PCR detection of cancer cell of S1 nuclease

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] 1) The experiment is divided into a normal cell control group (human bronchial epithelial HBE cells) and a tumor cell experimental group (human non-small cell lung cancer NCI-H460 cells). The cell culture of the two groups is the same as other experimental operating conditions. The theoretical length of the PCR amplified product is expected to be about 66bp.

[0049] 2) Subculture human non-small cell lung cancer NCI-H460 cells and human bronchial epithelial HBE cells to 6-well cell culture plates, respectively, in DMEM medium containing 10% calf serum (volume percentage) ((The medium solution contains 4.5 g / L D-glucose, 80mg / L glycine, 0.584g / L L-glutamine, 4mg / L pyridoxine hydrochloride, 110mg / L sodium pyruvate and 3.7g / L sodium bicarbonate, adjusted with hydrochloric acid pH value to 7.3)) in regular culture (95% humidity, 37°C, 5% CO 2 (Volume percentage) until the cell growth reaches 80% confluence, use PBS buffer to wash away the residual cell culture medium, then ad...

Embodiment 2

[0065] Other tumor cell proliferation testing cases:

[0066] 1) The experiment was divided into a normal cell control group (normal breast epithelial cells MCF-10A–parental, human embryonic kidney epithelial 293T cells) and a tumor cell experimental group (human breast cancer MCF-7 cells and human cervical cancer Siha cells), two groups The cell culture is the same as other experimental operating conditions. The theoretical length of the PCR amplified product is expected to be about 66bp.

[0067] 2) Subculture the MCF-10A-parental, MCF-7, 293T and Siha cells to a 6-well cell culture plate respectively, and conduct cell culture according to the method described in Example 1.

[0068] 3) Use the method described in Example 1 to set up a positive control and a negative control, while using Beta-Actin as an internal reference.

[0069] 4) The method described in Example 1 was used to perform experimental operations such as RNA extraction, reverse transcription PCR, S1 nuclease digestio...

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Abstract

The invention provides a DNA molecular probe; the molecular probe is DNA molecular probe of miRNA Let-7b; the DNA molecular probe has a stem ring structure. The DNA molecular probe is applied to detect miRNA in the cancer cell, the experimental result shows that miRNA Let-7b has low expression in non-small cell lung cancer, breast cancer cell, and cervical cancer cell, and has high expression in normal human pulmonary epithelial cell, breast cancer epithelial cell and human embryo kidney epithelial cell; the probe can simply and effectively detect the expression level of miRNA in the cancer cell.

Description

Technical field [0001] The invention relates to the field of biomedicine, in particular to a detection method and application of miRNA (microRNA) in tumor cells. Background technique [0002] miRNA is a single-stranded small RNA that is expressed endogenously in organisms, which is a type of non-coding RNA. It can regulate eukaryotic gene expression at the post-transcriptional level and translation level, thereby widely participating in eukaryotic development, cell differentiation and Physiological and pathological processes such as immune regulation and tumorigenesis. Abnormal expression of miRNA itself is an important factor in the occurrence and development of tumors. Therefore, the detection of miRNA expression levels in tumor cells is one of the important techniques for anti-tumor research. However, the length of mature miRNA is only about 20-24 nucleotides, and there is no polyadenylic acid tail. Therefore, the detection method of mRNA (messenger RNA) cannot be used, that ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6851C12N15/11C12Q1/6886
CPCC12Q1/6851C12Q1/6886C12Q2600/178C12Q2531/113C12Q2525/207C12Q2525/301C12Q2521/327
Inventor 曹春雨王发玲杨建林王艳林
Owner CHINA THREE GORGES UNIV
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