岭南现代临床外科2026，Vol.26Issue(4)：265-272,8.DOI:10.3969/j.issn.1009-976X.2026.04.009
用于肿瘤标志物发现的中等长度非编码RNA随机引物高通量测序方法开发与比较
Development and comparison of a random-primer high-throughput sequencing method for mid-sized non-coding RNA biomarker discovery in cancer
摘要
Abstract
Objective Precision surgery and individualized cancer treatment increasingly rely on molecular biomarkers that inform tumor classification,recurrence risk,prognosis,and therapeutic response.Mid-sized non-coding RNAs(mncRNAs)participate in ribosome biogenesis,RNA processing,and selective translation and have been associated with malignant progression and treatment response in multiple cancers,suggesting their potential as a source of novel tumor biomarkers.However,the complex secondary structures,nucleotide modifications,and diverse terminal configurations of mncRNAs make their detection highly susceptible to library-preparation bias,limiting systematic biomarker discovery.To address these challenges,we developed a random-primer DNA-adapter ligation sequencing method and compared it with two commonly used library-preparation strategies to evaluate its technical performance for mncRNA biomarker discovery in cancer.Methods HeLa cells were used as a tumor cell model and profiled using RNA-adapter ligation,poly(A)-tailing/template-switching,and the random-primer DNA-adapter ligation methods developed in this study.In the random-primer methods,an RNA adapter was ligated to the RNA 3′ end,followed by reverse transcription using a random-sequence-containing RT primer and SuperScript IV,and subsequent ligation of a DNA adapter to the cDNA.The resulting datas-ets were compared in terms of full-length RNA recovery,RNA length distribution,and RNA biotype composition.Results The three strategies exhibited distinct technical biases.RNA-adapter ligation yielded the lowest proportion of full-length RNA reads.Poly(A)-tailing/template-switching improved full-length RNA recovery to some extent but predominantly generated shorter RNA fragments.In contrast,the random-primer methods achieved the highest proportion of full-length RNA reads(approximately 40%)and substantially enhanced the detection of tRNA-related sequences.All three approaches detected diverse classes of mncRNAs,including rRNAs,snoRNAs,tRNAs,snRNAs,and miRNAs,while show-ing clear preferences for specific RNA biotypes,indicating that library preparation can directly influence the spectrum of candidate tumor biomarkers identified.Conclusion The random-primer methods devel-oped in this study showed advantages in recovering full-length RNAs and achieving broader coverage of tRNAs/tsRNAs,providing a new technical option for discovery-oriented studies of structurally complex and extensively modified mncRNAs.The method was validated using HeLa cells and should be further evaluated in surgical tumor specimens and blood-based samples from different solid tumors to establish its cross-cancer applicability and its ability to identify biomarkers associated with molecular stratifica-tion,recurrence,prognosis,and therapeutic response.关键词
肿瘤/中等长度非编码RNA/高通量测序/分子标志物/精准诊疗Key words
cancer/mid-sized non-coding RNA/high-throughput sequencing/molecular bio-marker/precision medicine分类
医药卫生引用本文复制引用
李好,张曼,邓锦思,占子凌,廖建友,郑琤..用于肿瘤标志物发现的中等长度非编码RNA随机引物高通量测序方法开发与比较[J].岭南现代临床外科,2026,26(4):265-272,8.基金项目
国家自然科学基金面上项目(82273033) （82273033）
