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System and method for quantitative detection of mRNA gene

A quantitative detection method and quantitative detection technology, applied in the field of quantitative detection of gene mRNA expression level, can solve the problems of environmental pollution, high experimental cost, and complicated experimental operation.

Inactive Publication Date: 2007-05-16
HUNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above methods have different research and application scopes according to their respective characteristics, but as quantitative detection techniques for gene expression, these methods have defects such as complex experimental operations, high experimental costs, poor repeatability of results, and easy environmental pollution. Difficulty with gene expression

Method used

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  • System and method for quantitative detection of mRNA gene
  • System and method for quantitative detection of mRNA gene

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Embodiment 1 (RNA detection selectivity analysis)

[0020] In 400μL reaction buffer (10mM Tris-HCl (pH8.0), 3mM MgCl 2 ) containing 25nM ING1 molecular beacon 5'-(TAMRA)-CGTTGATGGTTCCACTTCTCGTGCGTTCA ACG-(DABCYL)-3'.

[0021] Molecular beacon complementary DNA, random RNA sequence (70pM), molecular beacon complementary ING1RNA (70pM), incubate and hybridize at 37°C for 3-4 hours, then monitor the fluorescence intensity with a fluorometer F-2500, excitation wavelength 521nm, emission The wavelength is 578nm, and the fluorescent signal is detected (results are shown in Figure 2).

[0022] Figure 2 shows the fluorescent signals after hybridization of molecular beacons with different samples. It can be seen that the molecular beacons only hybridize with single-stranded complementary DNA and complementary RNA, causing an increase in fluorescent signal, but there is no change in fluorescent signal after hybridization with random sequence RNA. It shows that the method has hi...

Embodiment 2

[0023] Example 2: (Detection of the influence of gene transfection on ING1 gene expression)

[0024] After the transfection of ING1 gene, the MCF-7 tumor cell clone screened by G418 was expanded and cultured, and the total RNA of the cells was extracted; 2 ) containing 25nM molecular beacon 5'-(TAMRA)-CGTTGATGGTTCCACTTCTCGTGCGTTCAACG-(DAB CYL)-3', 3ug of total cellular RNA, incubated at 37°C for 4-6 hours, and then monitored the fluorescence with a fluorometer F-2500 Intensity, with an excitation wavelength of 521nm and an emission wavelength of 578nm, was used to detect the fluorescent signal (results shown in Figure 4), and the fluorescent signal value of the total RNA of MCF-7 cells hybridized with the molecular beacon was 89.88. According to the standard curve equation y (fluorescence value) = 1.9444x (RNA concentration) + 14.692, the ING1RNA concentration in the 400ul reaction system is calculated to be 38.66pM, and the ING1RNA content is 38.66×10 -12 ×400×10 -6 / 3ug=51...

Embodiment 3

[0025] Example 3 (Detection of Effects of Drug Treatment on ING1 Gene Expression)

[0026] Total cellular RNA was extracted using TRIzol kit; MCF-7 tumor cells not transfected with ING1 gene were treated with 100ug / ml anti-tumor drug 5-Fu for 12 hours and 24 hours, total cellular RNA was extracted, and mixed with 400 μL reaction buffer (10mM Tris-HCl (pH8.0), 3mM MgCl 2 ) containing 25nM molecular beacon 5'-(TAMRA)-CGTTGATGGTTCCACTTCTCGTGCGTTCA ACG-(DAB CYL)-3', 3ug of total cellular RNA, incubated at 37°C for 4-6 hours, and then monitored with a fluorescent instrument F-2500 Fluorescence intensity, the excitation wavelength is 521nm, the emission wavelength is 578nm, and the fluorescence signal is detected (results are shown in Figure 4).

[0027] The fluorescent signal value of total RNA extracted from MCF-7 treated for 12 hours after hybridization with molecular beacons was 51. According to the standard curve equation y (fluorescence value)=1.9444x (RNA concentration)+14....

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Abstract

The present invention relates to a quantitative analysis inspection technology of gene mRNA expression level. The inspection system consists of standard sample RNA, nucleic acid probe of molecular beacon and the RNA inspection sample in the corresponding cross breeding buffer solution. The present inspection technology can quantitatively inspect gene expression level of cells or tissue samples after subjecting to various treatments such as gene transfection, medicine treatment and gene interference. It has scientific application value in the field of life science and medical research.

Description

technical field [0001] The invention relates to the detection of genes, in particular to the quantitative detection of gene mRNA expression levels. Background technique [0002] With the development of life sciences, people began to study gene function from three levels: genome structure level, genome transcription level and protein level. At the level of gene transcription, the research on gene mRNA expression profile is mainly carried out. As the carrier of genetic information, gene mRNA can directly convert genetic information into protein. Since changes in gene transcription levels often lead to changes in many cellular events involved in cell survival, growth, and differentiation, quantitative detection of specific gene expression levels has become the core of gene function research. These research results have increasingly penetrated into the quantitative process design of tumor cell drug resistance factor expression regulation, chemotherapy response monitoring, gene-...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 王柯敏谭蔚泓刘斌李军王炜
Owner HUNAN UNIV