Method and kit for detecting small RNA

A kit and sequence technology, applied in the field of small RNA detection, can solve problems such as poor identification, and achieve the effects of strong specificity, high sensitivity and wide dynamic range

Inactive Publication Date: 2010-01-27
SHAOXING INST OF TECH COLLEGE OF ENG PEKING UNIV BIOENG CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

We know that usually single nucleotide mismatches in the middle position are easily recognized by C-probe ligation, but mismatches in the terminal positions may lead to poor discrimination
Furthermore, it remains a difficult task to simultaneously distinguish a group of highly similar miRNAs (such as the let-7 series) only through the specificity of the ligase itself.

Method used

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  • Method and kit for detecting small RNA
  • Method and kit for detecting small RNA

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preparation example Construction

[0043] 3. Preparation of dsDNA template. 50 pmol of DNA oligo was annealed by incubation at 75° C. for 5 minutes, and then slowly cooled to room temperature (approximately 30 minutes). The reaction to prepare the dsDNA template was in the presence of 10 mM Tris HCl (Tris HCl), 50 mM NaCl, 10 mM MgCl 2 , 1 mM dithiothreitol (DTT), 25 μM dNTPs, and 5UKlenow Fragment (3'-5'exo) (New England BioLabs, Eastwin Scientific, China) in a 20-μL volume at 37°C for one hour. The reaction mixture was then heated to 75°C for 20 min to inactivate the enzyme and slowly cooled to room temperature to anneal the dsDNA.

[0044] 4. In vitro transcription reaction. 20 μL of the above dsDNA template mixture was added to 30 μL containing 0.5 mM NTPs, 40 mM Tris-HCl, 6 mM MgCl 2 , 10mM DTT, 2mM spermidine, 40-200U ribonuclease inhibitor (Ribonuclease Inhibitor (TAKARA, China, Dalian)) and 50 U T7 RNA polymerase (New England BioLabs, EastwinScientific, China) in the in vitro transcription buffer, T...

Embodiment 1

[0053] Detection of let-7a. The nucleotide sequence of let-7a is shown in Table 2, and the nucleotide sequences of RT-primers and bridge probes used in the detection process, as well as primer 1 and primer 2 in branch amplification, are shown in Table 3. The test results are shown in figure 1 b. Depend on figure 1 B It can be seen that the horizontal axis of the branch amplification curve (the common logarithmic value of the initial miRNA number) and the vertical axis C T The values ​​showed a perfect linear relationship (R 2 = 0.997). As can be seen from the figure, the detection method of the present invention can detect as little as 10 3 (zeptomole) let-7a molecule and it has a dynamic range of at least 7 orders of magnitude for each reverse transcription reaction.

Embodiment 2

[0055] Detection of miR-1. The nucleotide sequence of mir-1 is shown in Table 2, and the nucleotide sequences of RT-primers, bridge probes used in the detection process, and primer 1 and primer 2 in branch amplification are shown in Table 3. The test results are shown in figure 2 . Depend on figure 2 It can be seen that the horizontal axis of the branch amplification curve (the common logarithmic value of the initial miRNA number) and the vertical axis C T There is a good linear relationship between the values ​​(R 2 = 0.992). As can be seen from the figure, the detection method of the present invention can detect as little as 10 4 mir-1 molecules with a dynamic range of at least 6 orders of magnitude for each reverse transcription reaction.

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Abstract

The invention relates to a method and a kit for detecting a small RNA, belonging to the field of detection of small RNAs. The method for detecting the small RNA comprises the following steps: 1. hybridizing an RT-primer and a target small RNA; 2. reversely transcribing; 3. connecting the head and the tail of a reversely transcribed DNA chain to form a ring by the mediation of a bridge probe; and 4. carrying out branch amplification real-time quantitative detection under the actions of DNA polyase, a first primer and a second primer. The method for detecting the small RNA has the advantages of high sensitivity, large dynamic range, high interference resistance, and the like and has wide application prospect in a plurality of fields of scientific experiment, clinical diagnosis, spot test, benchmark test, and the like relevant to the small RNA.

Description

technical field [0001] The invention relates to the detection field of small RNA. Background technique [0002] Small RNA is an important part of the noncoding RNA family (noncoding RNA, referred to as ncRNA), including microRNA (microRNA or miRNA), small interfering RNA (siRNA), small nuclear RNA, small sequence RNA, piwi protein-binding RNA (piRNA) Wait. Studies have found that small RNAs play a vital role in the development, growth, and differentiation of cells and tissues in organisms, and even the occurrence of diseases, as well as the invasion and defense of viruses. There are also various ways in which small RNAs function. They can directly regulate the expression of genes by modifying DNA, and can also regulate the amount of proteins by changing the stability of gene transcription products and so on. Among various types of small RNAs, microRNAs are the most important in function. [0003] MicroRNA is a non-coding RNA molecule with a length of 18-25 nucleotides in ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q2561/113C12N15/1096C12N15/10C12Q2525/307
Inventor 席建忠姚波李娟
Owner SHAOXING INST OF TECH COLLEGE OF ENG PEKING UNIV BIOENG CENT
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