Establishing method of circular RNA high-throughput sequencing library and kit thereof

A construction method and technology for sequencing libraries, which are applied in the field of circular RNA high-throughput sequencing library construction methods and kits, can solve the problems of high price and limited circular RNA research progress, and achieve low-cost, circular RNA The construction method of high-throughput sequencing library is efficient and the effect of low residue ratio

Active Publication Date: 2017-06-06
GUANGZHOU FOREVERGEN BIOTECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In addition, there is currently no special circular RNA library construction kit on the market, and researchers need to purchase RNA extraction, rRNA depletion, linear RNA depletion, library construction, etc. from QIAGEN, Illumina, Backman, NEB, Life technology, etc. products, and then explore suitable conditions to build a library
On the one hand, this combination of multiple reagents is expensive to build a library. On the other hand, it takes a long time for researchers with high experimental skills to explore the best experimental conditions, which seriously limits the progress of circular RNA research. Therefore, it is necessary to Develop a special kit that can efficiently and stably construct a circular RNA high-throughput sequencing library

Method used

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  • Establishing method of circular RNA high-throughput sequencing library and kit thereof
  • Establishing method of circular RNA high-throughput sequencing library and kit thereof
  • Establishing method of circular RNA high-throughput sequencing library and kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0118] Example 1 Construction of circular RNA high-throughput sequencing library in cell samples

[0119] (S1) Extraction of total RNA in cell samples

[0120] Take cell sample 1×10 7 Wash once with PBS at 4°C, then add 1mL Trizol to each well of the 6-well plate, and repeatedly pipette 10 times with a 1mL pipette tip. Collect the samples into EP tubes, add 200 μL of chloroform, mix up and down for 30 seconds and let stand for 3 minutes. Then centrifuge at 4° C. and 12,000 rpm for 15 minutes, and the solution lysate is divided into three layers, wherein the upper layer is RNA dissolved in the water phase. Take the upper layer solution and add an equal volume of isopropanol to it, mix well and let stand at 4°C for 10 min. After that, centrifuge again at 4° C. and 12000 rpm for 10 min and remove the supernatant. Then add 1 mL of 75% ethanol to the pellet and mix up and down to resuspend the pellet. Then centrifuge at 4° C. and 12000 rpm for 10 min, remove the supernatant, a...

Embodiment 2

[0172] Example 2 Construction of circular RNA high-throughput sequencing library in fresh liver cancer tissue samples

[0173] (S1) Extraction of total RNA in fresh liver cancer tissue samples

[0174] Take 50 g of fresh liver cancer tissue samples, wash once with PBS at 4°C, and cut the tissue into pieces. Then add 1mL Trizol and homogenize 15 times with a hand-held high-speed disperser. Afterwards, the samples were collected into EP tubes and 200 μL of chloroform was added, mixed up and down for 30 seconds, and then allowed to stand for 3 minutes. Then centrifuge at 4° C. and 12,000 rpm for 15 minutes, and the solution lysate is divided into three layers, wherein the upper layer is RNA dissolved in the water phase. Take the upper layer solution and add an equal volume of isopropanol to it, mix well and let stand for 10min. After that, centrifuge again at 4° C. and 12000 rpm for 10 min and remove the supernatant. Then add 1 mL of 75% ethanol to the pellet and mix up and d...

Embodiment 3

[0225] Example 3 Construction of circular RNA high-throughput sequencing library in FFPE samples

[0226] (S1) Extraction of total RNA in FFPE samples

[0227] Slice the FFPE sample into 8 μm thick slices, and immediately transfer 7 slices to a 1.5mL centrifuge tube. Then 1 mL of xylene was added and vortexed vigorously for 30 s, and centrifuged at 14000 rpm for 2 min. Then add 1 mL of ethanol to the sample and vortex for 30 s, then centrifuge at 14,000 rpm for 2 min, remove the supernatant, and keep the precipitate. Dry at 37 °C for 15 min to remove ethanol. Then add 200 μL lysate and 20 μl Proteinase K to the pellet and vortex to mix. This was followed by a water bath at a temperature of 55°C for 15 min, followed by a water bath at a temperature of 80°C for 15 min. After brief centrifugation at low speed, add 200 μL of buffer to the sample and vortex for 20 seconds. Then add 600 μL absolute ethanol to the sample and vortex mix for 20 s. Then transfer the mixture to an ...

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Abstract

The invention discloses an establishing method of a circular RNA high-throughput sequencing library and a kit thereof. The establishing method sequentially comprises the following steps: (S1) extracting total RNA from a sample; (S2) removing DNA in the sample; (S3) detecting and evaluating the quality of the total RNA in the sample, and determining that the RNA quality meets a requirement; (S4) removing rRNA; (S5) removing linear RNA; (S6) constructing the circular RNA library for high-throughput sequencing. The establishing method of the circular RNA high-throughput sequencing library, provided by the invention, is high in efficiency, stable, low in residual ratio of the rRNA, high in circular RNA detection efficiency, good in data reproducibility, and high in sequencing result verifying success rate, and is especially applicable to an FFPE tissue and other samples with poor RNA quality.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a method for constructing a circular RNA high-throughput sequencing library and a kit thereof. Background technique [0002] Circular RNA (ciucular RNA, circRNA) is a new member of the RNA family that is different from traditional linear RNAs. Coding RNA molecules. As early as 1980, circular RNA had been discovered, but for a long period of time, due to the limitation of research technology level, circular RNA was considered as a by-product formed by wrong variable splicing, which belongs to a kind of An extremely rare phenomenon, and even considered as a genetic accident or experimental human factors, it has not attracted the attention of the academic community. With the development of deep RNA sequencing and large-scale bioinformatics, researchers have discovered that there are a large number of circular RNA molecules in organisms. In 2012, Salzman et al. found t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/68C40B50/06
CPCC12N15/1093C12Q1/6806C40B50/06C12Q2535/122
Inventor 赖炳权罗景燕何重华何铭辉李伟琴唐毅黄鸿昌
Owner GUANGZHOU FOREVERGEN BIOTECH CO LTD
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