High-throughput sequencing library construction method for trace free RNA of body fluid

Abstract

The invention discloses a high-throughput sequencing library construction method for trace free RNA of body fluid. The method comprises the following steps: purifying a body fluid sample exRNA with asilicon substrate, performing dephosphorylation, adding polyA tails, and performing inverse transcription on tailing products by using template switching and nucleic acid locking technologies so as toobtain cDNA of which the 5'-end is provided with linkers; and performing low-cycle PCR (Polymerase Chain Reaction), and finally obtaining the high-throughput sequencing library. According to the method disclosed by the invention, by using the template switching and nucleic acid locking technologies, deep optimization is performed on problems encountered by trace RNA in the process of constructingthe high-throughput sequencing library, and the library construction time and cost can be greatly reduced. By using the technology in the invention, library construction can be completed on less than10ng of trace RNA in one day, high-quality data can be acquired by virtue of high-throughput sequencing, and many existing disease-related RNA biomarkers can be found by analysis.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for constructing a high-throughput sequencing library of small amounts of free RNA in body fluids. Background technique [0002] exRNA refers to extracellular RNA, including miRNA, Y-RNA, circRNA, lncRNA, etc. Compared with existing DNA and protein markers, RNA markers have better sensitivity, tissue specificity and diversity, bringing new expectations for better clinical testing. With the continuous advancement of technology, the use of trace RNA in serum or plasma to diagnose diseases has become a research direction that diagnostic medicine pays close attention to. This is because: 1) There are a large amount of RNA in serum or a rich variety. And these RNAs can exist relatively stably; 2) At the same time, we already know that RNA is involved in the expression regulation of various genes; 3) Blood is one of the most easily obtained diagnostic samples; 4) Moreover, RNA in...

AI Technical Summary

Problems solved by technology

The problems of the above methods are: 1) There is no screening ability for the size of exRNA, and sequences less than 15nt have no information value; the acquisition of many samples is limited, including volume (blood, saliva quantity) and morphology (FFPE, saliva, cerebrospinal fluid, etc. ), and the amount of available RNA is limited, so in many cases it is impossible to obtain more than 10ng of RNA; the method of connecting adapters is inefficient, and the amount of captured RNA is limited; 2) The library construction process is cumbersome and time-consuming, and only building The library takes 5 to 7 days; and the synthesis of the adapter sequence (adaptor) is expensive, and the overall library construction cost is high (>1200 yuan / sample)

The above problems limit the research progress and clinical application of humoral RNA.

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