Probe composition for hindering Globin mRNA reverse transcription, and application of probe composition

A composition and reverse transcription technology, applied in the direction of DNA/RNA fragments, recombinant DNA technology, biochemical equipment and methods, etc., can solve the problems of increasing the cost of detection, difficult to achieve efficient removal effect, reducing the detection efficiency of blood-derived RNA and success rate etc.

Pending Publication Date: 2021-09-10
YEASEN BIOTECHNOLOGY (SHANGHAI) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] The traditional olyA enrichment method, RNaseH probe digestion and removal method and biotin probe capture and removal method are difficult to achieve efficient rem...

Method used

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  • Probe composition for hindering Globin mRNA reverse transcription, and application of probe composition
  • Probe composition for hindering Globin mRNA reverse transcription, and application of probe composition
  • Probe composition for hindering Globin mRNA reverse transcription, and application of probe composition

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1: Design and preparation of Globin mRNA reverse transcription hindering removal probe

[0059]The Globin mRNA reverse transcription hindrance removal probe composition provided in the embodiment of the present invention can be applied to various high-throughput sequencing platforms, such as Illumina, Huada MGI-seq, Nanopore or Pacbio, etc., for transcriptomics and epigenetics The scope of detection in the field of transcriptomics research is used to effectively remove Globin mRNA from blood sample RNA.

[0060] The probe composition includes one or more combinations of single-stranded DNA probes designed and synthesized for the full-length sequence of Globin mRNA molecules. The probe sequences and modifications are shown in Table 1. Globin mRNA molecules include HBA1 mRNA (NM_000558.5) , HBA2 mRNA (NM_000517.6), HBB mRNA (NM_000518.5), HBG1 mRNA (NM_000559.3) and HBG2 mRNA (NM_000184.3) one or more combinations.

[0061] The length of each single-stranded DNA ...

Embodiment 2

[0068] Example 2: mRNA-seq sequencing verifies the effect of the Globin mRNA reverse transcription blocking probe composition on the removal of Globin mRNA in simulated blood sample RNA.

[0069] In this example, we used mRNA-seq sequencing to verify the effect of the Globin mRNA reverse transcription hindering probe composition on the removal of Globin mRNA in the prepared simulated blood total RNA sample (see schematic diagram figure 2 ).

[0070] The specific implementation is as follows:

[0071] 1) Preparation of simulated blood total RNA standard samples.

[0072] Globin cDNA sequence with T7 promoter at the 5' end and 30 adenosine nucleotides (polyA) at the 3' end was synthesized in vitro, and Globin mRNA was synthesized using Thermo Scientific's TranscriptAid T7 High Yield Transcription Kit (Cat#K0441) according to the instructions In vitro transcription synthesis.

[0073] Table 2

[0074] components Dosage Globin cDNA 1 μg 5× TranscriptAi...

Embodiment 3

[0113] Example 3: lncRNA-seq sequencing verifies the effect of Globin mRNA reverse transcription hindering probe composition on the removal of Globin mRNA in simulated blood sample RNA.

[0114] In this example, we used lncRNA-seq to verify the effect of the Globin mRNA probe mixture on the removal of Globin mRNA in simulated blood sample RNA (see schematic diagram Figure 7 ).

[0115] The specific implementation is as follows:

[0116] 1) rRNA depletion: 1 μg of simulated blood total RNA was depleted using Hieff NGS® MaxUp rRNADepletion Kit (Cat#12253) from Yisheng Biotechnology.

[0117]2) RNA library construction: The Hieff NGS® Ultima Dual-mode RNA Library PrepKit for Illumina (Cat#12252) of Yisheng Biotech was used to construct the RNA NGS library.

[0118] Table 4

[0119] components Dosage Ribosomal RNA was removed by the above method 4 μL 1 μM Globin probe mix xμL 2× Frag / Prime buffer 8.5 μL Add DEPC water to 17 μL

[01...

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Abstract

The invention provides a probe composition, which comprises 20 probes, for hindering Globin mRNA reverse transcription, and discloses application of the probe composition. According to the invention, reverse transcription of a target RNA is inhibited with high efficiency and high specificity during synthesis of one-chain cDNA by using a reverse transcription hindering probe method, so that the target RNA is hindered from participating in a downstream library building process. The probe can specifically recognize and bind to the human globulin RNA and hinder the reverse transcription of the globulin RNA so as to realize the purpose of removing the globulin RNA in the RNA library building process, 99.5% or above of Globin mRNA sequences in the blood sample RNA can be effectively removed in the reverse transcription process, and the probe has the advantages of being easy in operation (one step) and short in time consumption (2 min), and is very suitable for large-scale industrial automation.

Description

technical field [0001] The patent of the present invention relates to a probe composition for hindering Globin mRNA reverse transcription and its application, and belongs to the field of biotechnology. Background technique [0002] With the development and popularization of high-throughput sequencing technology, RNA sequencing has become an effective means for disease diagnosis and scientific research. [0003] However, RNA samples contain a large amount of ribosomal RNA (rRNA accounts for about 90%-95% of total RNA), which occupies a large amount of RNA sequencing data, resulting in a waste of sequencing data and an increase in cost. [0004] There are many rRNA removal methods on the market to increase the proportion of effective RNA sequencing data, such as polyA enrichment method, RNaseH probe digestion removal method, and biotin probe capture removal method. [0005] These methods have defects such as serious RNA loss, complex operation, and poor removal effect on low-...

Claims

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Application Information

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IPC IPC(8): C12Q1/6806C12N15/11
CPCC12Q1/6806C12Q2521/107C12Q2535/122C12Q2537/163
Inventor 江翱陈晶晶卢瑶王嫚孙睿曹振宋东亮
Owner YEASEN BIOTECHNOLOGY (SHANGHAI) CO LTD
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