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Recent data indicated that the next\u2010generation sequencing (NGS)\u2010based high\u2010throughput quantification of miRNAs from biofluids provided exciting possibilities for discovering biomarkers of various diseases and might help promote the development of the early diagnosis of cancer. However, the complex process of library construction for sequencing always introduces bias, which may twist the actual expression levels of miRNAs and reach misleading conclusions.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>We discussed the deviation issue in each step during constructing miRNA sequencing libraries and suggested many strategies to generate high\u2010quality data by avoiding or minimizing bias. For example, improvement of adapter design (a blocking element away from the ligation end, a randomized fragment adjacent to the ligation junction and UMI) and optimization of ligation conditions (a high concentration of PEG 8000, reasonable incubation temperature and time, and the selection of ligase) in adapter ligation, high\u2010quality input RNA samples, removal of adapter dimer (solid phase reverse immobilization (SPRI) magnetic bead, locked nucleic acid (LNA) oligonucleotide, and Phi29 DNA polymerase), PCR (linear amplification, touch\u2010down PCR), and product purification are essential factors for achieving high\u2010quality sequencing data. Moreover, we described several protocols that exhibit significant advantages using combinatorial optimization and commercially available low\u2010input miRNA library preparation kits.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusions<\/jats:title><jats:p>Overall, our work provides the basis for unbiased high\u2010throughput quantification of miRNAs. These data will help achieve optimal design involving miRNA profiling and provide reliable guidance for clinical diagnosis and treatment by significantly increasing the credibility of potential biomarkers.<\/jats:p><\/jats:sec>","DOI":"10.15302\/j-qb-021-0284","type":"journal-article","created":{"date-parts":[[2021,11,22]],"date-time":"2021-11-22T01:06:31Z","timestamp":1637543191000},"page":"321-332","source":"Crossref","is-referenced-by-count":1,"title":["The methodological challenge in high\u2010throughput profiling and quantifying microRNAs"],"prefix":"10.1002","volume":"10","author":[{"given":"Mengya","family":"Chai","sequence":"first","affiliation":[{"name":"College of Life Science and Technology Beijing University of Chemical Technology Beijing 100029 China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xueyang","family":"Xiong","sequence":"additional","affiliation":[{"name":"College of Life Science and Technology Beijing University of Chemical Technology Beijing 100029 China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Huimin","family":"Wang","sequence":"additional","affiliation":[{"name":"College of Life Science and Technology Beijing University of Chemical Technology Beijing 100029 China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lida","family":"Xu","sequence":"additional","affiliation":[{"name":"College of Life Science and Technology Beijing University of Chemical Technology Beijing 100029 China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2022,12]]},"reference":[{"key":"e_1_2_9_2_2","doi-asserted-by":"publisher","DOI":"10.1007\/s13238\u2010013\u20103001\u20105"},{"key":"e_1_2_9_3_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.jmb.2013.03.007"},{"key":"e_1_2_9_4_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2018.03.006"},{"key":"e_1_2_9_5_2","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkw116"},{"key":"e_1_2_9_6_2","doi-asserted-by":"publisher","DOI":"10.1038\/nrclinonc.2011.76"},{"key":"e_1_2_9_7_2","first-page":"1","volume-title":"Agilent Technologies","author":"Tissot C.","year":"2008"},{"key":"e_1_2_9_8_2","doi-asserted-by":"publisher","DOI":"10.5483\/BMBRep.2011.44.1.11"},{"key":"e_1_2_9_9_2","doi-asserted-by":"publisher","DOI":"10.1038\/nrg2504"},{"key":"e_1_2_9_10_2","doi-asserted-by":"publisher","DOI":"10.1080\/15476286.2016.1236172"},{"key":"e_1_2_9_11_2","doi-asserted-by":"publisher","DOI":"10.3390\/ijms14034934"},{"key":"e_1_2_9_12_2","doi-asserted-by":"publisher","DOI":"10.1007\/s40291\u2010016\u20100221\u20104"},{"key":"e_1_2_9_13_2","doi-asserted-by":"publisher","DOI":"10.4068\/cmj.2015.51.2.51"},{"key":"e_1_2_9_14_2","first-page":"37","article-title":"The Challenge of microRNA as a biomarker of epilepsy.","volume":"16","author":"Ma Y.","year":"2018","journal-title":"Curr. 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