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Circular small RNA library construction method and its application

A construction method and technology of single-stranded circular library, which are applied in the field of circular small RNA library construction

Active Publication Date: 2018-08-14
MGI TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the cPAL-based high-throughput sequencing platform does not have a corresponding small RNA library construction method. In order to solve this problem, the present invention has developed a small RNA with a length of only 18-30nt and constructed it into a single-stranded circular library, and technology that enables this library to be sequenced by the cPAL method

Method used

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  • Circular small RNA library construction method and its application
  • Circular small RNA library construction method and its application
  • Circular small RNA library construction method and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0111] 实施例1 小RNA单链环状文库的构建

[0112] 具体实验步骤(见 figure 1 中所示流程步骤):

[0113] 1.带有条形码(barcode)的3’接头的连接。该3’接头是一段DNA序列,与cPAL测序的锚定序列相匹配,并且带有10bp的条形码(barcode)序列,以便于区分不同的样品。在3’接头的5’端有腺苷酰化修饰,该修饰能够在没有三磷酸腺苷(ATP)的条件下被T4RNAligase 2truncated特异性识别,将其连接到RNA的3’羟基上,这样能够避免带有5’磷酸的RNA发生自连。具体的反应过程为:取1ug总RNA,加入1ul 10uM的3’接头,PCR仪中70℃反应2分钟,以打开序列的二级结构。再加入反应混合液:2N T4RNA ligase buffer5ul,RNase抑制剂(40U / ul)0.5ul,T4RNA ligase 2truncated(200U / ul)1ul,补无RNA酶水至反应体积为10ul。其中2N T4 RNA ligase buffer包括:100mMTris-HCl,20mM MgCl2,2mM DTT,25%的PEG8000,其余试剂为无RNA酶水。把反应混合液混匀后,在PCR仪中,25℃反应2小时。

[0114] 2.加入反转录引物与3’接头进行退火,以阻止过量的3’接头在下一步反应中与5’接头发生连接。由于3’接头是带有条形码(barcode)的,所加入的反转录引物必须与3’接头完全匹配,也要带有条形码(barcode),这样二者退火形成双链,过量的3’接头就不能被RNAligase识别并连接到5’接头上,减少了接头自连的形成。具体反应过程为:取0.5ul 100uM的带有条形码(barcode)的反转录引物加入加完3’接头的连接反应液中混匀,放入PCR仪中反应,反应程序为:75℃5min,37℃30min,25℃15min。

[0115] 3.5’接头的连接。5’接头是一段RNA序列,该序列同样与cPAL测序的锚定序列相匹配,其5’端和3’端未作特殊修饰,所以两端都是羟基。在ATP的存在下,使用T4RNA ligase 1能将RNA的5’磷酸基团与5’接头的3’羟基连接在一起。连接反应条件为:取1ul 10uM 5’接头,PCR仪中70℃反应2分钟,以打开...

Embodiment 2

[0132] Example 2 Identify the repeatability and stability of the construction method of the small RNA single-stranded circular library

[0133] The library preparation steps 1-8 in Example 1 were repeated, with the difference that two human cell RNA standards (UHRR and HBRR), mouse RNA and rice RNA were used as the source of total RNA. Each sample starts with 1ug of total RNA, first adds a 3' adapter, 16 samples are ligated with 3' adapters with different barcodes, and annealed with the corresponding reverse transcription primers with barcodes (barcode) to block Excess 3' adapters, religation of 5' adapters and reverse transcription and PCR amplification.

[0134] After the PCR reaction was completed, the 16 samples were divided into two groups, with 8 samples in each group (including 4 different RNA samples, each with one repetition), and then the two groups were mixed separately, and the samples were mixed with 6% acrylamide gel and gel respectively. 4% agarose gel electrop...

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Abstract

The invention specifically provides a method for constructing a single-stranded cyclic small RNA library. The single-stranded small RNA library may use a cPAL sequencing platform for sequencing. The application of the single-stranded small RNA library has the advantages of high sequencing throughput, high accuracy and simple operation.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular, the invention relates to a method for constructing a circular small RNA library and an application thereof. Background technique [0002] Small RNAs include several different types of noncoding RNAs: microRNAs (miRNAs), short interfering RNAs (siRNAs), small nucleolar RNAs (snoRNAs) and small nuclear RNAs (snRNAs). Among these endogenous small RNAs, miRNAs are the most comprehensively studied in terms of biological origin and functional mechanisms. miRNA is a kind of small RNA molecule with only 20-25 nucleotides, which mainly performs post-transcriptional regulation by binding to the target site on the mRNA, and then guides the degradation of the target mRNA or represses the translation of the target mRNA according to the degree of complementarity. It plays an important role in gene transcription, translation, cell growth and individual development. [0003] In recent years, the mea...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C40B50/06C40B40/06C12Q1/6806
Inventor 祝珍珍张春燕耿春雨章文蔚蒋慧
Owner MGI TECH CO LTD