Ultra-trace small RNA (Ribonucleic Acid) sequencing method

Through Ultra-PANDORA-Seq technology, a one-tube reaction and AlKB are used to modify the enzyme activity reaction, and the small RNA library is directly built using cell lysate, solving the problem that the existing technology cannot accurately analyze the small RNA types except miRNA and piRNA, and achieving efficient and accurate detection of small cellular RNAs.

CN119709977APending Publication Date: 2025-03-28SHANGHAI FIRST MATERNITY & INFANT HOSPITAL
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Patent Information

Application Number
CN202411980078.4
Authority / Receiving Office
CN · China
Patent Type
Applications(China)
Current Assignee / Owner
Filing Date
2024-12-31
Publication Date
2025-03-28

AI Technical Summary

Technical Problem

Existing small RNA sequencing technologies cannot accurately analyze other small RNA types except miRNA and piRNA in cells, such as rsRNA and tsRNA, and the detection accuracy is insufficient in microcell samples.

Method used

An ultra-trigram small RNA sequencing method, called Ultra-PANDORA-Seq, is used to perform RNA terminal repair and AlKB demodification enzyme activity reaction through a one-tube reaction step by step expansion system. The cell lysate is directly used to build small RNA libraries, reducing the RNA extraction and separation steps, and using ferrous ions in the cell to assist the AlKB enzyme activity reaction.

Benefits of technology

It has achieved efficient and accurate detection of small RNAs in microcellular, overcome the technical difficulties of traditional methods in microcellular, and provided feasible and reliable analytical means for in-depth research on the functional mechanism of small RNAs.

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Abstract

The invention relates to an ultramicro small RNA (Ribonucleic Acid) sequencing method, which adopts a tubular reaction, carries out RNA terminal repair and AlKB demodification enzyme activity reaction by expanding a system step by step, and comprises cell lysis, RNA terminal repair, 3'adapter connection, AlKB demodification enzyme activity reaction, RNA purification, RT (Reverse Transcription) annealing, 5 'adapter connection, cDNA (Complementary Deoxyribose Nucleic Acid) reverse transcription, library amplification and NGS (Next Generation System). According to the present invention, the improvement is performed on the basis of the existing PANDORA-Seq technology, the efficient and accurate detection of the trace cell small RNA is achieved by optimizing the reaction conditions, and the UltraTra-PANDORA-Seq method is successfully established. According to the method, the technical problem of the traditional method in trace cell small RNA detection is solved, and a feasible and reliable analysis means is provided for deep research on the functional mechanism of the small RNA in a living body. The development of the Ultra-PANDORA-Seq technology provides important scientific basis and technical support for exploration of intergenerational genetic mechanisms of metabolic diseases, early clinical diagnosis, development of small RNA drugs and clinical intervention.
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