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Construction method of dated bloodstain miRNA high-throughput sequencing library

A sequencing library and construction method technology, applied in the field of molecular biology, can solve the problems of high amount of miRNA templates, inability to apply trace blood stains, no reports on miRNA library construction methods and sequencing methods, etc., and achieve the effect of increasing lysis time

Active Publication Date: 2018-12-11
BEIJING CENT FOR PHYSICAL & CHEM ANALYSIS
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  • Abstract
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AI Technical Summary

Problems solved by technology

Currently, miRNA extraction and library construction methods are aimed at fresh blood samples and tissue cells, etc., and require a high amount of miRNA templates, usually requiring more than 100ng of total RNA, which cannot be applied to trace blood stains
At present, there are no reports on miRNA library construction and sequencing methods for forensic samples such as old bloodstains

Method used

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  • Construction method of dated bloodstain miRNA high-throughput sequencing library
  • Construction method of dated bloodstain miRNA high-throughput sequencing library
  • Construction method of dated bloodstain miRNA high-throughput sequencing library

Examples

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Embodiment 1

[0101] Example 1 Construction method of old bloodstain miRNA high-throughput sequencing library

[0102] 1. miRNA extraction

[0103] In this example, Tiangen miRNA extraction kit was used for miRNA extraction.

[0104] 1. Take 5cm 2 Old bloodstains (placed for half a year) shredded to 3-5mm 2 , add 200 μl volume of lysate MZ, let it stand at room temperature for 10 minutes, shake and mix with an oscillator for 30 seconds.

[0105] 2. Leave at room temperature for 5 minutes to completely separate the nucleic acid-protein complex.

[0106] 3. Centrifuge at 12,000 rpm for 10 minutes at room temperature, take the supernatant, and transfer it to a new RNase-free centrifuge tube.

[0107] 4. Add 200 μl chloroform, cover the tube cap, shake vigorously for 15 sec, and place at room temperature for 5 min.

[0108] 5. Centrifuge at 12,000 rpm at room temperature for 15 minutes. The sample will be divided into three layers: a yellow organic phase, an intermediate layer and a colorl...

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Abstract

The invention discloses a construction method of a dated bloodstain miRNA high-throughput sequencing library. The method comprises the steps of pretreatment of a dated bloodstain sample, extraction ofmiRNA and construction of a miRNA sequencing library, wherein the library construction includes: (1) connection of miRNA 5' and 3' junctions; (2) reverse transcription; (3) PCR (Polymerase Chain Reaction) amplification; (4) agarose gel electrophoresis on a PCR amplification product, gel cutting and recovery of fragments of 145 to 160 bp to obtain the sequencing library. By adopting the method disclosed by the invention, construction of the dated bloodstain miRNA library can be finished within 48 hours, and only a 5cm<2> dated bloodstain (containing about 10 to 100ng RNA) sample is needed at least. Reuse of dated forensic evidence is facilitated, the reuse of dated forensic evidence appears in the field of forensic medicine for the first time, and strong technical support is provided for expanding the application of miRNA in forensic medicine.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a method for constructing an old bloodstain miRNA high-throughput sequencing library. Background technique [0002] Individual identification is an important task of forensic evidence, and it is used to reveal individual identity, investigate and solve cases, and an important link in compensation. Traditional individual identification methods are based on genomic DNA short tandem repeats (Short Tandem Repeats, STRs), single nucleotide polymorphisms (Single Nucleotide Polymorphisms, SNPs) analysis and so on. These methods have high requirements on the amount and integrity of templates, and when the amount of templates is too small or degraded, the accuracy of the detection results will be affected. At the same time, it is also impossible to judge the tissue, body fluid source and individual characteristics of the sample. [0003] Early individual identification used the method o...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/6806C40B50/06
CPCC12N15/1096C12Q1/6806C40B50/06C12Q2531/113C12Q2525/191C12Q2565/125C12Q2535/122
Inventor 方晨武会娟严江伟刘旭钱嘉林刘文丽张小莉
Owner BEIJING CENT FOR PHYSICAL & CHEM ANALYSIS
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