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Method of constructing pre-miRNA 5'RACE-seq library in plants

A plant and library technology, applied in the field of constructing pre-miRNA5'RACE-seq library in plants, can solve problems such as library construction, RNA ligase inoperability, and linker addition

Inactive Publication Date: 2017-06-09
SHENZHEN UNIV
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

The reason is that pre-miRNA has a stem-loop structure compared with mature miRNA, and the 3' end protrudes, which makes ordinary RNA ligase unable to work, and it is difficult to add adapters at the 5' end, so the library construction cannot be completed by ordinary methods

Method used

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  • Method of constructing pre-miRNA 5'RACE-seq library in plants
  • Method of constructing pre-miRNA 5'RACE-seq library in plants
  • Method of constructing pre-miRNA 5'RACE-seq library in plants

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Embodiment 1

[0018] Step 1, extraction of total plant RNA

[0019] 1. Take 100mg of fresh plant tissue in a clean 1.5mL centrifuge tube.

[0020] 2. Quick-freeze the plant tissue in liquid nitrogen and crush it with a gun tip.

[0021] 3. Add 1mL TRIZOL and mix upside down, and let it stand at room temperature for 5min.

[0022] 4. Add 200 μL of chloroform, mix by inversion, and let stand at room temperature for 15 minutes.

[0023] 5. Centrifuge at 12000X g for 15 minutes, and absorb 400 μL of supernatant.

[0024] 6. Add 400 μL of isopropanol, mix evenly by inversion, and let stand at -20°C for 30 minutes to precipitate RNA.

[0025] 7. Centrifuge at 12,000X g for 15 minutes at 4°C and discard the supernatant.

[0026] 8. Add 1 mL of 75% ethanol to wash the RNA, centrifuge at 12000X g for 5 min at 4°C.

[0027] 9. Dry RNA at room temperature, add 30 μL ddH 2 O dissolves RNA.

[0028] 10. Store the extracted RNA at -80°C.

[0029] Step 2, RNA preparation (50-400)

[0030] 1. Prep...

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Abstract

The invention provides a method of constructing pre-miRNA 5'RACE-seq library in plants; by using the method, it is possible to clearly visualize the variation of a base at pre-miRNA 5' end in plants. Through RNA extraction, joint adding, reverse transcription, looping, PCR (polymerase chain reaction) and the like, the technical difficulty in adding a joint to the 5' end is solved successfully; the novel method is provided to construct pre-miRNA 5'RAC-seq library for high-throughput sequencing. The method employs SDS-PAGE (sodium dodecyl sulfate and polyacrylamide gel electrophoresis) RNA separating technique, 3'-RACE joint technique, single-chain DNA cyclizing technique and other techniques. The looping technique is introduced to the construction process of pre-miRNA library, the joint adding problem is solved, and a high-quality pre-miRNA 5'RACE-seq library can be constructed.

Description

technical field [0001] The invention relates to a method for constructing a pre-miRNA 5' RACE-seq library in plants. Background technique [0002] With the development of sequencing technology, the sequencing of transcriptome and degradome is becoming more and more common. As the basis of next-generation sequencing technology, library construction plays a decisive role in the sequencing results. Although the construction of mRNA libraries is well established, in plants, the construction of pre-miRNA libraries has not been reported. Pre-miRNA is the precursor of miRNA, and its modification is crucial for the formation of mature miRNA. Therefore, it is of great biological significance to ascertain the nucleotide changes of pre-miRNA for the study of miRNA formation. At present, the methods for constructing mRNA and miRNA libraries are mature, but in plants, the methods for constructing pre-miRNA5'RACE-seq libraries are still blank. The reason is that pre-miRNA has a stem-loo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/68C40B50/06
CPCC12N15/1096C12Q1/6806C40B50/06C12Q2521/107C12Q2525/191C12Q2525/207C12Q2531/113
Inventor 宋剑波刘琳岳路明莫小为杨海奇陈雪梅莫蓓莘
Owner SHENZHEN UNIV
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