Ribosome-RNA-removed reverse transcription primer pool, kit and method for removing ribosome RNA

A technology of reverse transcription primers and ribosomes, applied in DNA/RNA fragments, recombinant DNA technology, chemical libraries, etc., can solve the problems of data redundancy, high price, affecting the detection and analysis of mRNA or LncRNA, etc. The effect of application range

Pending Publication Date: 2020-08-14
广东美格基因科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Existing kits for rRNA removal and transcriptome library construction, mainstream kits use RNA probes combined with streptavidin magnetic beads to remove rRNA, such as Illumina’s Stranded Total RNA LT-(with Ribo-Zero TM Plant), the probes of its kit are exogenously synthesized, expensive, and can only be designed based on 1-2 species, other species or even subspecies have not been The removal efficiency can be guaranteed, and the initial amount of total RNA is 100ng-1ug. For samples with an initial amount of total RNA of 1ng-100ng, there is no kit that can effectively remove rRNA and construct a transcriptome library.
This limitation may lead to serious rRNA residues and data redundancy in the construction of transcriptome sequencing libraries other than those marked by the kit, or in the construction of transcriptome sequencing libraries from low-input samples, which will affect the subsequent detection and analysis of mRNA or LncRNA

Method used

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  • Ribosome-RNA-removed reverse transcription primer pool, kit and method for removing ribosome RNA
  • Ribosome-RNA-removed reverse transcription primer pool, kit and method for removing ribosome RNA
  • Ribosome-RNA-removed reverse transcription primer pool, kit and method for removing ribosome RNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] 1. Reverse transcription primer design and rRNA removal experiment

[0062] 1. Plant cultivation

[0063] The cooperative laboratory cultivates and collects Arabidopsis plant tissues through tissue culture.

[0064] 2. Total RNA extraction

[0065] Follow the instructions of Qiagen's QIAamp RNA RNeasy Mini Kit for total RNA extraction, starting with 1 μg of total RNA for subsequent reactions.

[0066] 3. Mix reverse transcription primers into a reverse transcription primer pool

[0067] Design and synthesis of reverse transcription primers. The reverse transcription is designed on the RNA sequence of rRNA (NC_003071.7) according to the design density of 500bp / strand, 1000bp / strand and 2000bp / strand. The mixed reverse transcription primer is RT Primer pool, Each reverse transcription primer was diluted to 10μM with sterile water.

[0068] Design density of 1000bp / bar (denoted as RT-1000bp group), corresponding to the pool of 5 reverse transcription primers that are ticked in 1Kbp / i...

Embodiment 2

[0107] Example 2 Exploring the concentration of reverse transcription primers

[0108] 1. Plant cultivation: the specific method is the same as in Example 1.

[0109] 2. Total RNA extraction: The specific method is the same as in Example 1, and the initial amount of total RNA is 1 μg.

[0110] 3. Mixing of reverse transcription primers

[0111] The reverse transcription primers are designed for reverse transcription with a design density of 1000bp / strand. The mixed reverse transcription primers are RT Primerpool, and each reverse transcription primer is diluted with sterile water to 5μM, 10μM, 50μM and 100μM. Corresponding to the pool of 5 reverse transcription primers that are ticked in the 1000bp / item in Table 1, each reverse transcription primer is complementary to a region of the precursor RNA sequence. The concentration of any two reverse transcription primers in the reverse transcription primer pool is equal. The reverse transcription primers were synthesized by Shanghai Jieru...

Embodiment 3

[0123] Example 3 Exploration of RNase H and DNase I dosage

[0124] 1. Plant cultivation: the specific method is the same as in Example 1.

[0125] 2. Total RNA extraction: The specific method is the same as in Example 1, and the initial amount of total RNA is 1 μg.

[0126] 3. Mixing of reverse transcription primers

[0127] The reverse transcription primers are designed for reverse transcription with a design density of 1000 bp / strand. The mixed reverse transcription primers are RT Primerpool, and each reverse transcription primer is diluted to 10 μM with sterile water. Corresponding to the pool of 5 reverse transcription primers that are ticked in the 1000bp / item in Table 1, each reverse transcription primer is complementary to a region of the precursor RNA sequence. The concentration of any two reverse transcription primers in the reverse transcription primer pool is equal. The reverse transcription primers were synthesized by Shanghai Jierui Biological Engineering Co., Ltd.

[0...

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Abstract

The invention discloses a ribosome-RNA-removed reverse transcription primer pool, a kit and a method for removing ribosome RNA. The sequences of the reverse transcription primer pool are shown as SEQID NO: 1-8; and the kit comprises the reverse transcription primer pool. The method comprises the following steps: designing reverse transcription primers covered by different densities by utilizing the rRNA sequences shared by plants; according to the difference of rRNA sequences in RNA which needs to be removed specifically, performing specific reverse transcription to obtain cDNA sequences which are completely and reversely complementary with the target RNA; specifically digesting rRNA and cDNA of a heterozygous strand through subsequent RNase H and DNase I; and finally removing the ribosome RNA. The method can be matched with a library building kit to finally obtain a transcriptome library with high enough concentration, so that the wide application of transcriptome sequencing in the fields of basic research, clinical diagnosis, drug research and development and the like is effectively realized.

Description

Technical field [0001] The present invention relates to the field of biotechnology, and more specifically, to a reverse transcription primer pool and a kit for removing ribosomal RNA to construct a transcription group sequencing library, and a method for removing ribosomal RNA. Background technique [0002] With the development of modern science and technology, life science research has entered the age of omics. Gene and genome sequencing technology has become an indispensable method in modern life science research, especially genomics research. In recent years, the rapid development of next-generation genome sequencing technology has brought unprecedented prosperity in genomics research. Next-generation sequencing technology has been widely used in various fields of life sciences, agriculture, medicine, environmental protection, and forensics. With the rapid development of next-generation high-throughput sequencing technology, transcriptome sequencing has become an important m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C40B50/06
CPCC12N15/11C40B50/06
Inventor 曹亮吴胜标喻志红蒋华束文圣
Owner 广东美格基因科技有限公司
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