Phospho-small RNA-seq reveals circulating, extracellular mRNA/lncRNAs as potential biomarkers in human plasma: PNK evaluation on synthetic, equimolar pool [SYNTHETICPNK]

Extracellular RNAs (exRNAs) in blood and other biofluids have attracted great interest as potential biomarkers in liquid biopsy applications, as well as for their potential biological functions. Whereas it is well-established that extracellular microRNAs are present in human blood circulation, the degree to which messenger RNAs (mRNA) and long noncoding RNAs (lncRNA) are represented in plasma is less clear. Here we report that mRNA and lncRNA species are present as small fragments in plasma that are not detected by standard small RNA-seq methods, because they lack 5'-phosphorylation or carry 3'-phosphorylation. We developed a modified sequencing protocol (termed “phospho-sRNA-seq”) that incorporates upfront RNA treatment with T4 polynucleotide kinase (which also has 3' phosphatase activity) and compared it to a standard small RNA-seq protocol, using as input both a pool of synthetic RNAs with diverse 5' and 3' end chemistries, as well exRNA isolated from human blood plasma. This series uses the synthetic pools of small RNAs to demonstrate the efficacy of phospho-sRNA-seq to enable capture of sRNAs lacking a 5' phosphate and/or having a 3' phosphate. Overall design: Two TruSeq smallRNA-seq libraries were prepared from a synthetic, equimolar pool of 476 small RNAs (15 - 90 nt), containing oligos with different end chemistries: 5' P + 3'OH (n=352), 5'OH + 3'P (n=60), 3'OH + 5'OH (n=60), 5'P + 3' 2OME (n=4). Libraries were generated by 1) directly sequencing the pool or 2) pre-treating the pool with T4 Polynucleotide Kinase (PNK).