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A method and composition for removing ribosomal nucleic acid rRNA in a total rna sample

A ribosome and sample technology, applied in DNA/RNA fragments, recombinant DNA technology, biochemical equipment and methods, etc., can solve the problems of removal efficiency not higher than 80%, unsatisfactory removal effect, waste and other problems

Active Publication Date: 2019-11-12
承启医学(深圳)科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Existing commercial kits, such as Ribo-Zerogold (Epicentre), Ribo-Minus TM (Thermo Fisher Scientific), although they can remove most of the rRNA, the cost of the removal method based on magnetic beads is relatively high, and the removal effect is not ideal, especially for small rRNA, such as 5S and 5.8S rRNA, the removal efficiency is not high More than 80%, these residual rRNA often also occupy a large amount of data capacity in the analysis, resulting in great waste
Secondly, the above method has high requirements on the quality of the total RNA of the sample. If the total RNA of the sample is slightly degraded, the rRNA removal effect will be greatly affected.

Method used

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  • A method and composition for removing ribosomal nucleic acid rRNA in a total rna sample
  • A method and composition for removing ribosomal nucleic acid rRNA in a total rna sample
  • A method and composition for removing ribosomal nucleic acid rRNA in a total rna sample

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Embodiment 1

[0053] Example 1: Method principle for efficiently removing ribosomal rRNA from total RNA of samples based on hybridization degradation method

[0054] The present invention provides a specific method for removing ribosomal rRNA, comprising the following steps, such as figure 1 shown.

[0055] 1. For different species of the sample, design probes for the full-length sequences of all rRNAs of the species, and mix the probes.

[0056] 2. Add the probe mixture to the total RNA of the sample, slowly anneal, and the probe will specifically hybridize with the target rRNA to form a DNA:RNA hybrid duplex;

[0057] 3. Add RNase H enzyme-specific degradation probes and rRNA-bound hybrid double strands;

[0058] 4. DNase mixed enzyme removes trace DNA in the sample and degrades the remaining DNA probe mixture, and terminates the reaction;

[0059] 5. Purify the sample RNA after removing rRNA.

[0060] This method can be widely used for efficient removal of rRNA from total RNA in samp...

Embodiment 2

[0061] Embodiment 2: the design of DNA probe

[0062] For different species of samples, the method of the present invention detects the full-length sequences of all rRNAs of different species.

[0063] In this example, DNA probes for removing human rRNA (including 28S rRNA, 18S rRNA, 5.8S rRNA, 5S rRNA, 12S rRNA, and 16S rRNA) were designed. The specific method is as follows:

[0064] Obtain the full-length sequence of human rRNA from NCBI, 5S rRNA (accession number: NR_023379), 5.8S rRNA (accession number: NR_003285), 12S rRNA (accession number: NC_012920.1(648-1601)), 16S rRNA (accession number : NC_012920.1(1671-3229)), 18S rRNA (accession number: NR_003286), 28S rRNA (accession number: NR_003287).

[0065] 5S rRNA (121bp), 5.8S rRNA (159bp), 12S rRNA (954bp), 16S rRNA (1559bp), 18S rRNA (1969bp), 28S rRNA (5025bp) in human RNA, the total length of these rRNA full-length sequences is 9787bp, For the full-length sequences of these rRNAs, a single-stranded DNA probe of 50 ...

Embodiment 3

[0066] Embodiment 3: obtain DNA probe sequence

[0067] Design and prepare single-stranded DNA probes, the specific sequences are as follows:

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[0085] All probe molecules were mixed in equal proportions to form a probe mixture, and the concentration of each probe in the probe mixture was 5 μg.

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Abstract

The invention relates to a method, composition and kit for removal of ribosomal nucleic acid rRNA from a total RNA sample.

Description

technical field [0001] The invention relates to a method, composition and kit for removing ribosomal nucleic acid rRNA in a total RNA sample. Background technique [0002] Since the abundance of rRNA is very high, accounting for 80% to 95% of the total RNA, its presence will complicate the analysis of other related RNA molecules in the sample. Among the problems posed by rRNA is the analysis of fragmented related RNA molecules. For example, transcriptome microarray analysis and transcriptome sequencing will generate a large part of redundant data, resulting in a lot of waste. If rRNA can be efficiently removed, the effective utilization of data can be greatly improved. Especially in the analysis of non-3'poly A tail RNA and non-coding RNA, rRNA must be removed, otherwise the data availability of these genes is very poor. [0003] At present, the technical principle of removing rRNA from the total RNA of the sample is basically based on magnetic beads with streptavidin gro...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6816C12N15/11
CPCC12Q1/6816C12Q2521/327
Inventor 赵盼盼钟嘉泳张弓杜高飞赵晶金静洁
Owner 承启医学(深圳)科技有限公司
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