Method for extracting microRNA precursor cDNA from cDNA library synthesized from small RNA

An isolation and precursor technology, applied in the field of molecular biology, can solve the problems of difficult to find miRNA, low cloning effect, and many repeated sequences, and achieve the effect of improving cloning and sequencing efficiency and good separation effect.

Inactive Publication Date: 2010-12-15
NANJING AGRICULTURAL UNIVERSITY
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Problems solved by technology

[0004] The common limitation of finding miRNAs by cloning sequencing is: due to the large number of siRNAs (small interfering RNAs) in the small RNA library, the cloning effect is greatly reduced, and it is difficult for miRNAs with low-level expression, or in a spec...

Method used

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  • Method for extracting microRNA precursor cDNA from cDNA library synthesized from small RNA
  • Method for extracting microRNA precursor cDNA from cDNA library synthesized from small RNA
  • Method for extracting microRNA precursor cDNA from cDNA library synthesized from small RNA

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Embodiment 1

[0017] 1. Small RNA extraction

[0018] A Roche small RNA (<100bp) extraction kit (High Pure miRNA Isolation Kit, Cat. No. 05 080 576 001) was used. The specific operation steps are as follows:

[0019] 1) Add 400 μL 20% binding buffer (Binding Buffer) to a 2 mL empty test tube;

[0020] 2) Add 1-50 mg of muscle tissue sample to the test tube, and break the tissue with a homogenizer;

[0021] 3) Centrifuge the test tube at 14,000 rpm for 2 minutes at 4°C, and then take about 150-188 μL of the supernatant into another new 1.5 mL test tube;

[0022] 4) Add 312μL of Binding Buffer and shake for 3×5s;

[0023] 5) Assemble the filter tube and collection tube in the kit, and suck the mixed solution into the filter tube above;

[0024] 6) Centrifuge at 13000×g for 30s, and collect the liquid in the collection tube below;

[0025] 7) Add 200 μL of Binding Enhancer to the collection tube and shake for 3×5 s;

[0026] 8) Assemble the filter tube and collection tube in the kit, and...

Embodiment 2

[0246] Using this method to study porcine miRNA, the cDNA single strands in two different regions in the gel were separated and recovered, and then cloned and sequenced after recovery to double strands. After the sequencing results were analyzed with BioEdit and RNAfold software, it was found that the ratios of hairpin sequences in the sequencing results of clones in A and B regions were significantly different. Among the 63 sequencing results in area A, almost all of the sequences had a "clover" type secondary structure, and none of them basically met the hairpin type secondary structure standard of miRNA precursors. This shows that most of the small RNAs in this region are not miRNA precursors. In the 45 sequencing results in area B, a total of 5 sequences were found that basically conformed to the secondary structure of the miRNA precursor hairpin, but most of the other sequences still did not meet the definition requirements of the miRNA precursor hairpin secondary structu...

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Abstract

The invention discloses a method for extracting a microRNA precursor cDNA from a cDNA library synthesized from small RNA, which comprises the following steps of: carrying out the PCR amplification on the cDNA; carrying out the PCR amplification on the obtained cDNA fragments by using a primer with the 5' end with biotin labels, carrying out degeneration on the cDNA double strands obtained by amplification, and carrying out the cDNA double-strain separation by using a magnetic bead technology; renaturing the cDNA single strands obtained by separation to form single-strain cDNA with different secondary structures and respectively recovering, carrying a PCR reaction to form double strands; and carrying out enzyme digestion on PCR fragments by restriction enzymes, and cloning and sequencing. The invention can effectively enhance the efficiency of cloning and sequencing miRNA.

Description

technical field [0001] The invention relates to a method for isolating a microRNA precursor cDNA from a cDNA library synthesized from small RNA, in particular to a single-stranded cDNA library separation technology based on the secondary structure characteristics of the microRNA precursor, belonging to the field of molecular biology. Background technique [0002] microRNA (miRNA) is a kind of non-coding single-stranded RNA molecule encoded by endogenous genes with a length of about 22 nucleotides. It is a single-stranded RNA precursor with a hairpin structure of about 70-90 bases Produced after Dicer enzyme processing. The current preferred method for laboratory identification of microRNA (miRNA) is the sequencing analysis of size-separated cDNA libraries. The original kit was used to clone small interfering RNA molecules of about 22nt, but it seems to work on endogenous small RNA molecules as well. Thus, many such kits are also used to clone miRNAs. Kits for cloning smal...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 周波刘红林
Owner NANJING AGRICULTURAL UNIVERSITY
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