Dataset Information

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

ABSTRACT:

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.

SUBMITTER: Liu Z

PROVIDER: S-EPMC9299576 | biostudies-literature | 2022 Jul

REPOSITORIES: biostudies-literature

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Publications

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

Liu Zhongzhen Z Wang Taifu T Yang Xi X Zhou Qing Q Zhu Sujun S Zeng Juan J Chen Haixiao H Sun Jinghua J Li Liqiang L Xu Jinjin J Geng Chunyu C Xu Xun X Wang Jian J Yang Huanming H Zhu Shida S Chen Fang F Wang Wen-Jing WJ

Clinical and translational medicine 20220701 7

<h4>Background</h4>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.<h4>Methods</h4>Here, we developed polyadenylation ligation-mediated sequencing (PALM-Seq), an RNA sequencing strategy employing treatment of RNA with T4 polynucleotide kinase to ...[more]

PMID: 35858042

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Project description:BackgroundRecent advances in sequencing technology have opened a new era in RNA studies. Novel types of RNAs such as long non-coding RNAs (lncRNAs) have been discovered by transcriptomic sequencing and some lncRNAs have been found to play essential roles in biological processes. However, only limited information is available for lncRNAs in Drosophila melanogaster, an important model organism. Therefore, the characterization of lncRNAs and identification of new lncRNAs in D. melanogaster is an important area of research. Moreover, there is an increasing interest in the use of ChIP-seq data (H3K4me3, H3K36me3 and Pol II) to detect signatures of active transcription for reported lncRNAs.ResultsWe have developed a computational approach to identify new lncRNAs from two tissue-specific RNA-seq datasets using the poly(A)-enriched and the ribo-zero method, respectively. In our results, we identified 462 novel lncRNA transcripts, which we combined with 4137 previously published lncRNA transcripts into a curated dataset. We then utilized 61 RNA-seq and 32 ChIP-seq datasets to improve the annotation of the curated lncRNAs with regards to transcriptional direction, exon regions, classification, expression in the brain, possession of a poly(A) tail, and presence of conventional chromatin signatures. Furthermore, we used 30 time-course RNA-seq datasets and 32 ChIP-seq datasets to investigate whether the lncRNAs reported by RNA-seq have active transcription signatures. The results showed that more than half of the reported lncRNAs did not have chromatin signatures related to active transcription. To clarify this issue, we conducted RT-qPCR experiments and found that ~95.24% of the selected lncRNAs were truly transcribed, regardless of whether they were associated with active chromatin signatures or not.ConclusionsIn this study, we discovered a large number of novel lncRNAs, which suggests that many remain to be identified in D. melanogaster. For the lncRNAs that are known, we improved their characterization by integrating a large number of sequencing datasets (93 sets in total) from multiple sources (lncRNAs, RNA-seq and ChIP-seq). The RT-qPCR experiments demonstrated that RNA-seq is a reliable platform to discover lncRNAs. This set of curated lncRNAs with improved annotations can serve as an important resource for investigating the function of lncRNAs in D. melanogaster.

Dataset Information

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

Background

Methods

Results

Conclusion

Publications

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

Similar Datasets

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