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Method for simply and quickly preparing ready-to-use esiRNA

A DNA probe and polymer block technology, applied in the field of small RNA preparation, can solve problems affecting the preparation and application of esiRNA

Inactive Publication Date: 2012-07-18
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the preparation of esiRNA in the prior art is a rather complicated process, including at least 6 steps: PCR amplification, transcription, enzyme digestion, purification, esiRNA quantification and normalization ( figure 1 ), these steps include the need to transfer, purify, quantify, and normalize liquid samples, requiring the use of expensive instruments such as liquid handling systems, centrifuges, and spectrometers
These tedious and expensive work have seriously affected the large-scale preparation and application of esiRNA

Method used

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  • Method for simply and quickly preparing ready-to-use esiRNA
  • Method for simply and quickly preparing ready-to-use esiRNA
  • Method for simply and quickly preparing ready-to-use esiRNA

Examples

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preparation example Construction

[0032] 1. Preparation of polymer blocks:

[0033] Acrylamide (acrylamide:bisacrylamide=19:1) 10wt% glycerol solution, 0.1M sodium phosphate buffer solution (pH=7.0), 3wt% methacrylic acid, 0.005wt% APS and 2mM TEMED was mixed well under a nitrogen atmosphere, then transferred to test tubes and incubated overnight at 65°C to allow polymerization.

[0034] 2. Immobilization of DNA probes on polymer blocks:

[0035] Use MES (0.1M) buffer solution (pH=6.0) containing EDC (2mM) and sulfo-NHS (2mM) to activate the carboxyl group on the polyacrylamide polymer block, and then add the 5'-terminal amino-modified DNA Oligomer (Invitrogen Co. Ltd., Shanghai) and incubated overnight at room temperature. The sequence of the DNA probe used in the PCR amplification reaction (that is, the probe immobilized on the first nucleic acid chip) is as follows: 5'-amino-TCACAGGATGGCTAATACGACTCACTATAGGGC-3', and the DNA probe used in the transcription process (that is, the immobilized The sequence of...

Embodiment 1

[0051] The bottom of the 96-well plate commonly used in the laboratory was pierced to form small holes, and a capillary was inserted into each small hole and fixed with PDMS, and polyacrylamide microspheres of the same volume and size were fixed at the lower end of the capillary. The first nucleic acid chip and the second nucleic acid chip can be obtained by immobilizing different DNA probes on polyacrylamide, wherein the DNA probe sequence on the first nucleic acid chip can be complementary to a sequence of the PCR amplification product DNA molecule, and the second The DNA probe sequence on the two-nucleic acid chip can be complementary to a sequence of transcription product RNA. Immerse the first nucleic acid chip into the PCR amplification solution to carry out the PCR amplification reaction. After one PCR reaction, take out the chip and rinse it, and then add another set of PCR amplification solutions that do not contain DNA templates to carry out the PCR amplification reac...

Embodiment 2

[0055] The experimental process is the same as in Example 1, except that the materials for preparing polymer microspheres are changed to polymethacrylamide, polylactic acid (PLA), polylactic acid-glycolic acid polymer (PLGA), polyacrylic acid (PAA), polyformaldehyde Acrylic acid, poly 2-hydroxyethyl (meth) acrylate, poly N-isopropyl (meth) acrylamide, polyvinyl acetate or polyacrylamine, the results obtained are similar to those of Example 1.

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Abstract

The invention relates to a preparation method of a small RNA (Ribose Nucleic Acid), in particular to a new method for simply and quickly preparing ready-to-use esiRNA at a low cost. The method has the beneficial effects that a chip integrating a macromolecule microsphere (macromolecule block) is adopted to simply and quickly prepare the esiRNA at a low cost and is utilized for amplification, transcription and enzymic digestion on a polymer bracket, so that liquid transfer, purification and other operation steps are simplified; the preparation method of the esiRNA is reduced from the existing 6 steps to 3 steps provided by the invention, so that the preparation process of the esiRNA is greatly simplified; and meanwhile, the chip is utilized to cut and normalize the quantity of the esiRNA product, so that the esiRNA can be directly used for the research of loss-of-function caused by gene silencing without further treatment.

Description

technical field [0001] The present invention relates to a preparation method of small RNA, in particular to a simple, fast and low-cost new method for preparing ready-to-use esiRNA. Background technique [0002] RNA interference (RNA interfering, RNAi) is a phenomenon in which double-stranded RNA (double-stranded RNA, dsRNA) induces efficient and specific degradation of homologous mRNA. It is an ancient and evolutionarily highly conserved gene expression regulation mechanism in the biological world. In 1998, researchers discovered that injecting dsRNA into C.elegans or Drosophila can specifically inhibit gene expression, and called this phenomenon of inhibiting specific gene expression triggered by dsRNA as RNAi. Subsequently, the basic research and application of RNAi quickly became one of the hot areas of life science. RNAi research has made a breakthrough, and was rated as one of the top ten scientific advances in 2001 by the "Science" magazine, and ranked first in the t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 席建忠黄璜
Owner PEKING UNIV
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