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

Area of Science:

• Biochemistry
• Molecular Biology
• Genomics

Background:

• Cell-free messenger RNA (cf-mRNA) and long non-coding RNA (cf-lncRNA) are crucial biomarkers in liquid biopsy for disease prediction, diagnosis, and prognosis.
• Simultaneous characterization of coding and non-coding RNAs in human biofluids presents a significant analytical challenge.

Purpose of the Study:

• To develop and validate a novel RNA sequencing strategy for the simultaneous quantification and characterization of cell-free RNAs (cfRNAs).
• To explore the potential of cfRNAs as biomarkers across various human biofluids.

Main Methods:

• Developed polyadenylation ligation-mediated sequencing (PALM-Seq), an RNA sequencing strategy.
• Utilized T4 polynucleotide kinase for 5' phosphate and 3' hydroxyl generation in cfRNA fragments.
• Employed RNase H treatment to deplete abundant RNA species, enabling comprehensive cfRNA profiling.

Main Results:

• Successfully identified differentially abundant mRNA, lncRNA, and microRNA in pregnant women's blood plasma.
• Characterized cfRNAs across multiple biofluids (blood plasma, saliva, urine, seminal plasma, amniotic fluid), revealing body fluid-specific variations in RNA biotypes.
• Observed that cf-mRNA profiles reflect tissue of origin, with immune cells being a major source in blood plasma and saliva.
• Found short RNA fragments (<50 nt) to be predominant in most biofluids, while seminal plasma and amniotic fluid showed a tendency to retain longer RNA fragments.
• Identified distinct 3' pyrimidine and 5' adenine preferences in cf-mRNA and cf-lncRNA, correlated with fragment length.

Conclusions:

• PALM-Seq enables the simultaneous characterization of cf-mRNA and cf-lncRNA.
• This advancement aids in elucidating cfRNA biology and promotes their clinical application in liquid biopsy.