Polyadenylation ligation‐mediated sequencing (PALM‐Seq) characterizes cell‐free coding and non‐coding RNAs in human biofluids

• Published in: Clinical and translational medicine Vol. 12; no. 7; pp. e987 - n/a
• Main Authors: Liu, Zhongzhen, Wang, Taifu, Yang, Xi, Zhou, Qing, Zhu, Sujun, Zeng, Juan, Chen, Haixiao, Sun, Jinghua, Li, Liqiang, Xu, Jinjin, Geng, Chunyu, Xu, Xun, Wang, Jian, Yang, Huanming, Zhu, Shida, Chen, Fang, Wang, Wen‐Jing
• Format: Journal Article
• Language: English
• Published: United States John Wiley & Sons, Inc 01.07.2022; John Wiley and Sons Inc; Wiley
• Subjects: Alzheimer's disease; Amniotic fluid; biofluids; Biopsy; Cancer; Cell-Free Nucleic Acids - genetics; cell‐free RNA; Clinical medicine; Female; Humans; in vitro diagnosis; Medical diagnosis; MicroRNAs - genetics; Plasma; Polyadenylation - genetics; Pregnancy; RNA sequencing; RNA, Long Noncoding - genetics; RNA, Messenger - genetics; Sequence Analysis, RNA; Urine; Womens health; United Kingdom > UK; England; RNA sequencing; in vitro diagnosis; biofluids; cell-free RNA
• ISSN: 2001-1326; 2001-1326
• DOI: 10.1002/ctm2.987

Background Cell‐free messenger RNA (cf‐mRNA) and long non‐coding RNA (cf‐lncRNA) are becoming increasingly important in liquid biopsy by providing biomarkers for disease prediction, diagnosis and prognosis, but the simultaneous characterization of coding and non‐coding RNAs in human biofluids remains challenging. Methods Here, we developed polyadenylation ligation‐mediated sequencing (PALM‐Seq), an RNA sequencing strategy employing treatment of RNA with T4 polynucleotide kinase to generate cell‐free RNA (cfRNA) fragments with 5′ phosphate and 3′ hydroxyl and RNase H to deplete abundant RNAs, achieving simultaneous quantification and characterization of cfRNAs. Results Using PALM‐Seq, we successfully identified well‐known differentially abundant mRNA, lncRNA and microRNA in the blood plasma of pregnant women. We further characterized cfRNAs in blood plasma, saliva, urine, seminal plasma and amniotic fluid and found that the detected numbers of different RNA biotypes varied with body fluids. The profiles of cf‐mRNA reflected the function of originated tissues, and immune cells significantly contributed RNA to blood plasma and saliva. Short fragments (<50 nt) of mRNA and lncRNA were major in biofluids, whereas seminal plasma and amniotic fluid tended to retain long RNA. Body fluids showed distinct preferences of pyrimidine at the 3′ end and adenine at the 5′ end of cf‐mRNA and cf‐lncRNA, which were correlated with the proportions of short fragments. Conclusion Together, PALM‐Seq enables a simultaneous characterization of cf‐mRNA and cf‐lncRNA, contributing to elucidating the biology and promoting the application of cfRNAs. PALM‐Seq simultaneously characterizes the abundance and molecular signature of cell‐free coding and non‐coding RNAs. The profiles and end motifs of cf‐mRNA and cf‐lncRNA can distinguish biofluids. Seminal plasma tends to retain longer fragments than other biofluids. Pyrimidine dominates at the 3′ ends of cf‐mRNA and cf‐lncRNA fragments, and its frequency is correlated with the fragment length.