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Reverse transcription primer pool for removing rRNA, kit and method for removing rRNA

A technology of reverse transcription primers and ribosomes, applied in biochemical equipment and methods, DNA/RNA fragments, DNA preparation, etc., can solve data redundancy, affect the detection and analysis of mRNA or LncRNA, rRNA residues, etc. To achieve the effect of broadening the scope of application

Pending Publication Date: 2020-08-11
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

The probes of the kits of the above two principles are all exogenously synthesized, expensive, and can only be designed based on 1 or 2 species. The removal efficiency of other species cannot be guaranteed, and the initial amount of total RNA 100ng~1ug, for samples whose total RNA input amount is 1ng~100ng, there is no kit that can effectively remove the rRNA and construct the transcriptome library
This limitation may lead to other eukaryotic species other than the species marked in the kit, or serious rRNA residues and data redundancy in the construction of transcriptome sequencing libraries from samples such as plasma, serum and Exosome, which will affect the subsequent mRNA or LncRNA identification. Detection and analysis

Method used

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  • Reverse transcription primer pool for removing rRNA, kit and method for removing rRNA
  • Reverse transcription primer pool for removing rRNA, kit and method for removing rRNA
  • Reverse transcription primer pool for removing rRNA, kit and method for removing rRNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0062] 1. Cell culture

[0063] Use GIBCO's DMEM and 10% FBS serum at 37°C, 5% CO 2 The 293T cells were cultured under culture conditions, and after 2 days of normal culture, the cell density was about 60-80% (40,000-50,000).

[0064] 2. Total RNA extraction

[0065] Total RNA was extracted according to Qiagen's QIAamp RNA RNeasy Mini Kit instructions, starting with 1 μg of total RNA for subsequent reactions.

[0066] 3. The reverse transcription primers are mixed into a reverse transcription primer pool

[0067] Design and synthesis of reverse transcription primers, design reverse transcription primers on the precursor RNA 45S sequence of rRNA (sequence number: NR_046235.1) according to the design density of 500bp / , 1000bp / , 2000bp / and 5000bp / product , the mixed reverse transcription primer is RT Primer pool (abbreviated as reverse transcription primer pool), and each reverse transcription primer...

Embodiment 2

[0107] The concentration exploration of embodiment 2 reverse transcription primers

[0108] 1. Cell culture: the specific method is the same as in Example 1.

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

[0110] 3. Reverse transcription primer mix

[0111] The reverse transcription primers were designed for reverse transcription at a design density of 1000bp / strip. The mixed reverse transcription primers were RT Primerpool, and each reverse transcription primer was diluted to 5 μM, 10 μM, 50 μM and 100 μM with sterile water. A pool formed by mixing a total of 12 reverse transcription primers corresponding to all ticked 1000bp / entry items in Table 1, each reverse transcription primer is complementary to a region on the precursor RNA 45S sequence. The concentrations of any two reverse transcription primers in the reverse transcription primer pool are equal. The reverse transcription primers ...

Embodiment 3

[0124] Embodiment 3 RNase H and DNase I dosage exploration

[0125] 1. Cell culture: the specific method is the same as in Example 1.

[0126] 2. Extraction of total RNA: the specific method is the same as in Example 1, and the initial amount of total RNA is 1 μg.

[0127] 3. Reverse transcription primer mix

[0128] The reverse transcription primers were designed for reverse transcription at a design density of 1000bp / strip. The mixed reverse transcription primers were RT Primerpool, and each reverse transcription primer was diluted to 10 μM with sterile water. A pool formed by mixing a total of 12 reverse transcription primers corresponding to all ticked items of 1000 bp / item in Table 1, each reverse transcription primer is complementary to a region on the precursor RNA 45S sequence. The concentrations of any two reverse transcription primers in the reverse transcription primer pool are equal. The reverse transcription primers were synthesized by Shanghai Jierui Bioengine...

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Abstract

The invention discloses a reverse transcription primer pool for removing rRNA, a kit and a method for removing the rRNA. The sequences of the reverse transcription primer pool are as shown in SEQ ID NO:1-21. The kit comprises the reverse transcription primer pool. Reverse transcription primers covered with different densities are designed by using rRNA sequences shared by eukaryotes; specific reverse transcription is performed according to the difference of the rRNA sequences in RNA needing to be removed specifically, so that cDNA sequences completely and reversely complementary with target RNA are obtained; rRNA and cDNA of a hybrid chain are specifically digested through subsequent RNase H and DNase I; and finally the rRNA is removed. According to the method, a library kit can be established and finally a transcriptome library with the sufficiently high concentration is obtained, thereby effectively implementing wide application of transcriptome sequencing in the fields of basic research, clinical diagnosis, drug research and development and the like.

Description

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

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806C12N15/10C40B50/06C12N15/11
CPCC12Q1/6806C12N15/1093C40B50/06C12Q2521/327C12Q2521/345C12Q2521/107
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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