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Oligonucleotide micro-array chip for detecting small molecule RNA

A microarray chip and antisense oligonucleotide technology, applied in the biological field, can solve the problems such as the difference and interference of miRNA abundance that cannot be accurately reflected, and achieve the effects of large dynamic range, low cost and high sensitivity of detection.

Inactive Publication Date: 2006-03-08
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Claims
  • Application Information

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Problems solved by technology

However, the cDNA generated by reverse transcription cannot accurately reflect the difference in miRNA abundance, especially the use of random primers is more likely to cause many interferences

Method used

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  • Oligonucleotide micro-array chip for detecting small molecule RNA
  • Oligonucleotide micro-array chip for detecting small molecule RNA
  • Oligonucleotide micro-array chip for detecting small molecule RNA

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example 1

[0023] 11 antisense oligonucleotides (such as figure 2 A) and a negative control (Negat.Ctrl.) that is not complementary to known miRNAs were used to make miRNA detection microarray chips. The RNA fraction enriched with small molecular weight was first oxidized with sodium periodate, and then reacted with biotin-X-hydrazide (purchased from Sigma). After the reaction, the biotin-labeled RNA was precipitated with ethanol. The labeled RNA was hybridized with the detection chip in a hybridization solution (50% formamide prehybridization / hybridization solution at 25° C. for 18 hours), and then reacted with streptavidin-coupled quantum dots. After washing (in 1×SSC / 0.5% SDS at 37° C. for 10 minutes), scan with ScanArray 5000, and then perform normalization analysis with QuantArray. The result is as figure 2 Shown in B and C.

example 2

[0025]The labeled RNA obtained in step (1) was hybridized with the detection chip in the hybridization solution, and then reacted with colloidal gold (purchased from Sigma) coupled with streptavidin (colloidal gold was diluted 1:5, and 10 Microliter reacted with the chip, room temperature for 1 hour), followed by silver enhancement for 20 minutes. Take pictures with a common digital camera after washing, then analyze with QuantArray, the results are as follows: image 3 shown.

[0026] From figure 2 and image 3 It can be seen from the results that both methods give the same miRNA expression profile, and both have good repeatability. From Figure 4 It can be seen from the results that the detection limit of the miRNA microarray using the labeling method of the present invention can reach 39 pmol. Furthermore, the inventors believe that under optimal conditions the limit of detection can be lower.

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Abstract

This invention provides a method for displaying miRNA expression spectrum including: 1, processing a combined miRNA antisense oligonucleotide probe to a micro-array chip 2, oxidizing RNA molecular 3' end in the sample to a double-aldehyde group then to mark for the miRNA molecules by the hydrazide label molecules, 3, intercrossing the sample in step 1 and the micro-array chip in step 1 to test said mark. This invention also provides a reagent box for testing the miRNA.

Description

technical field [0001] The present invention relates to the field of biotechnology. More specifically, the present invention relates to a method for detecting small RNA and a kit for the method. Background technique [0002] MicroRNA (microRNA, hereinafter referred to as "miRNA") is a kind of non-coding RNA with a size of about 22 nucleotides, which regulates the expression of its target mRNA by specifically recognizing the target messenger RNA (mRNA). In different tissues, organs and different developmental stages, the expression profile of miRNA is different, and it may play an important regulatory role in various biological development and physiological activities. The misexpression of miRNA can lead to diseases such as cancer (He & Hannon, Nat Rev. Genet. 5 (2004): 522-531). The study of miRNA expression profile will play a huge role in promoting the study of miRNA biological function. [0003] The existing studies on miRNA expression profiles mostly use Northern hybr...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 阮康成金由辛梁汝强李洋李建勋
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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