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Method for detecting small molecule RNA chip by ELISA

An enzyme-linked immunosorbent assay and chip technology, which can be used in biochemical equipment and methods, measurement devices, analytical materials, etc., and can solve problems such as limiting the wide application of miRNA chips.

Inactive Publication Date: 2009-08-05
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] In summary, most of the existing miRNA chips are based on fluorescent labeling technology, and the detection of chips requires expensive fluorescent scanners, which limits the wide application of miRNA chips, especially in clinical testing.

Method used

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  • Method for detecting small molecule RNA chip by ELISA
  • Method for detecting small molecule RNA chip by ELISA
  • Method for detecting small molecule RNA chip by ELISA

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Experimental program
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preparation example Construction

[0055] The preparation method of miRNA chip is known in the art, and generally, miRNA chip comprises conventional solid-phase support (being substrate), and the oligonucleotide probe that is positioned at sample area on substrate, and described probe can pass through for example Covalently immobilized on the solid support. The spotting probe may be an oligonucleotide sequence complementary to any kind of miRNA or an oligonucleotide sequence corresponding to the miRNA sequence. As a preferred mode of the present invention, the probes on the miRNA chip contain sequentially from 5' to 3': amino-(A) n - a nucleotide sequence complementary to miRNA; wherein n=5-20 (preferably 8-12, eg 10). Described solid phase carrier is exactly the substrate material that has oligonucleotide probe on it, and common carrier includes: glass flake, plastic flake, nitrocellulose (NC) membrane, PDVF membrane etc., wherein particularly preferred It's a glass slide.

[0056] Based on the ELISA techno...

Embodiment 1

[0070]Example 1 Preparation of rat miRNA chip

[0071] Material

[0072] Glass slides and (3-glycidyloxypropyl)-trimethoxysilane [(3-glycidyloxypropyl)-trimethoxysilane, GOPTS] were purchased from Sigma. Avidin-coupled alkaline phosphatase was purchased from R&D Company, and BCIP / NBT was purchased from Calbiochem Company. DNA probes were synthesized at Invitrogen. The RNA extraction reagent was RNArose from Huashun Company. The rats used in the experiment were 9-week-old male SD rats purchased from Shanghai Experimental Animal Center.

[0073] equipment

[0074] 0lympus C-4000Z digital camera, ordinary microscope. The biochip spotting instrument is PixSys 5500, a product of Cartesian Company, and the assembly spotting needle is SMP4, a product of TeleChem Company. Laser confocal biochip scanner is Genepix 4000B.

[0075] chip making

[0076] Standard 25mm×75mm glass slides were activated according to the method described in Liang, R.Q. et al., Colorimetric detection of...

Embodiment 2

[0080] Example 2 Obtaining and labeling of rat miRNA

[0081] Use RNArose to extract total RNA from the epididymis of 9-week-old SD rats frozen in liquid nitrogen according to the instructions, electrophoresis on 15% polyacrylamide urea denaturing gel, and recover the polypropylene located between the 18nt and 31nt DNARuler by slicing the gel under ultraviolet light Amide glue. The small fragments of RNA were recovered by dipping, precipitated with ethanol, and stored at -20°C. The biotinylation of small RNA was carried out according to the method described in Liang, R.Q. et al., An oligonucleotide microarray formicroRNA expression analysis based on labeling RNA with quantum dot and nanogold probe. Nucleic Acids Res, 2005(33), e17: from 90 μg total RNA The obtained 18 μl of 0.5 μg / μl small molecule RNA was diluted with 9 μl of 0.25M pH 5.6 sodium acetate buffer and 4 μl of DEPC water, added 5 μl of 5 mM sodium periodate solution, and reacted at 25°C for 90 minutes in the dark...

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Abstract

The invention belongs to the field of biotechnology, and discloses a method for detecting a miRNA chip through the enzyme linked immunosorbent assay. The enzyme-linked immune technology is firstly applied to the detection of miRNA in the method, and the method has high detection sensitivity and can detect the data of the chip through a common digital camera, a microscope or a scanner, thereby greatly reducing the cost of miRNA chip detection.

Description

technical field [0001] The invention belongs to the field of biotechnology, and more specifically, the invention relates to a method for detecting a small molecule RNA (miRNA) chip by an enzyme-linked immunosorbent method. Background technique [0002] In recent years, microRNA as a broad class of regulatory genes has been gradually known. microRNA widely exists in multicellular organisms, and plays an important regulatory role in many biological processes such as developmental regulation, cell apoptosis, cell proliferation, and emergency response. Faced with a wide variety of miRNAs, gene chips are playing an increasingly important role in the research of miRNAs due to their advantages such as trace, rapid, and large-scale parallel detection. [0003] At present, miRNA detection technology mainly adopts miRNA expression profile detection technology, and commonly used research methods are miRNA cloning and Northern hybridization. Not only can new miRNAs be discovered by cl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N33/535
Inventor 阮康成李巍
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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