Non-coding RNAs in stroke pathology, diagnostics, and therapeutics.

Ischemic stroke is a leading cause of death and disability worldwide. Methods to alleviate functional deficits after ischemic stroke focus on restoration of cerebral blood flow to the affected area. However, pharmacological or surgical methods such as thrombolysis and thrombectomy have a narrow effective window. Harnessing and manipulating neurochemical processes of recovery may provide an alternative to these methods. Recently, non-coding RNA (ncRNA) have been increasingly investigated for their contributions to the pathology of diseases and potential for diagnostic and therapeutic applications. Here we will review several ncRNA - H19, MALAT1, ANRIL, NEAT1, pseudogenes, small nucleolar RNA, piwi-interacting RNA and circular RNA - and their involvement in stroke pathology. We also examine these ncRNA as potential diagnostic biomarkers, particularly in circulating blood, and as targets for therapeutic interventions. An important aspect of this is a discussion of potential methods of treatment delivery to allow for targeting of interventions past the blood-brain barrier, including lipid nanoparticles, polymer nanoparticles, and viral and non-viral vectors. Overall, several long non-coding RNA (lncRNA) discussed here have strong implications for the development of pathology and functional recovery after ischemic stroke. LncRNAs H19 and ANRIL show potential as diagnostic biomarkers, while H19 and MALAT1 may prove to be effective therapeutics for both minimising damage as well as promoting recovery. Other ncRNA have also been implicated in ischemic stroke but are currently too poorly understood to make inferences for diagnosis or treatment. Whilst the field of ncRNAs is relatively new, significant work has already highlighted that ncRNAs represent a promising novel investigative tool for understanding stroke pathology, could be used as diagnostic biomarkers, and as targets for therapeutic interventions.

A method for simultaneous detection of small and long RNA biotypes by ribodepleted RNA-Seq.

RNA sequencing offers unprecedented access to the transcriptome. Key to this is the identification and quantification of many different species of RNA from the same sample at the same time. In this study we describe a novel protocol for simultaneous detection of coding and non-coding transcripts using modifications to the Ion Total RNA-Seq kit v2 protocol, with integration of QIASeq FastSelect rRNA removal kit. We report highly consistent sequencing libraries can be produced from both frozen high integrity mouse hippocampal tissue and the more challenging post-mortem human tissue. Removal of rRNA using FastSelect was extremely efficient, resulting in less than 1.5% rRNA content in the final library. We identified > 30,000 unique transcripts from all samples, including protein-coding genes and many species of non-coding RNA, in biologically-relevant proportions. Furthermore, the normalized sequencing read count for select genes significantly negatively correlated with Ct values from qRT-PCR analysis from the same samples. These results indicate that this protocol accurately and consistently identifies and quantifies a wide variety of transcripts simultaneously. The highly efficient rRNA depletion, coupled with minimized sample handling and without complicated and high-loss size selection protocols, makes this protocol useful to researchers wishing to investigate whole transcriptomes.