Method for constructing circRNA high-throughput sequencing library of FFPE sample

A construction method and sequencing library technology, which is applied in the field of formalin-fixed paraffin-embedded sample circRNA high-throughput sequencing library construction, can solve the problems of cumbersome and complicated steps, and achieve simple operation steps, short library construction time, Avoid the effect of large sample requirements

Inactive Publication Date: 2018-09-18
广州密码子基因科技有限公司
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Problems solved by technology

The steps of this method are cumbersome and complicated, and exogenous linear RNA and exogenous circular RNA nee

Method used

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  • Method for constructing circRNA high-throughput sequencing library of FFPE sample
  • Method for constructing circRNA high-throughput sequencing library of FFPE sample
  • Method for constructing circRNA high-throughput sequencing library of FFPE sample

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Embodiment 1

[0042] Example 1 Construction of circRNA high-throughput sequencing library

[0043] Embodiments of the present invention take human tissue paraffin section RNA as a sample, such as figure 1 As shown, the specific steps are as follows:

[0044] Step (1): Extract total RNA from paraffin sections.

[0045] The thickness of the paraffin section is 10 μm.

[0046]Add 1mL xylene to the centrifuge tube containing the slices for dewaxing, shake vigorously for 10s, incubate at 60°C for 5min, centrifuge at 14000rpm at room temperature for 5min, discard the supernatant, repeat once; then add 1.2mL absolute ethanol, shake and mix well, Centrifuge at 14,000 rpm for 5 min, repeat once, discard the supernatant, and air dry for 5 min; use the QIAGEN miRNeasy FFPE Kit extraction reagent, follow the kit instructions for subsequent RNA extraction, and dissolve the obtained total RNA in deionized water to remove nucleases.

[0047] Use Qubit 3.0 Fluorometer (Life Technology) to measure the ...

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Abstract

The invention discloses a method for constructing a circRNA high-throughput sequencing library of an FFPE (formalin-fixed and paraffin-embedded) sample. The method concretely comprises the following steps: (1) extracting total RNA from paraffin sections; (2) adding a PolyA end to the sample RNA; (3) capturing and separating linear RNA by Oligo dT magnetic beads; (4) further removing residual linear RNA; and (5) constructing the circRNA high-throughput sequencing library. The FFPE sample RNA has the characteristics of damages, low content and poor integrity, and the method for constructing thecircRNA high-throughput sequencing library of the FFPE sample has the advantages of simplicity in operation, short time, low cost, stableness in obtaining the library meeting high-throughput sequencing requirements, and avoiding of the problems of large sample size, high rRNA removal cost and low efficiency in the traditional library constructing methods.

Description

technical field [0001] The invention relates to a method for constructing a high-throughput sequencing library, belonging to the fields of biotechnology and molecular biology, and in particular to a method for constructing a circRNA high-throughput sequencing library for formalin-fixed paraffin-embedded (FFPE) samples. Background technique [0002] In recent years, the incidence of cancer has increased significantly, seriously threatening human health, and tumor research has become a global hotspot. Although tumor research methods and research perspectives are constantly updated, human tumor tissue specimens are still an indispensable and important object for tumor research. Formalin-Fixed and Parrffin-Embedded (FFPE) is a tissue processing and preservation method commonly used in pathology departments. FFPE tissue samples are simple to prepare, cheap to manufacture, and easy to store. They are widely preserved in hospitals and tissue sample banks around the world. They are...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/6806C12Q1/6869C40B50/06
CPCC12Q1/6806C12N15/1013C12Q1/6869C40B50/06C12Q2535/122
Inventor 丁向明孙萍刘明李杰申健
Owner 广州密码子基因科技有限公司
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