Neurodegeneration
This project will use this novel technology to profile circRNAs in AD brain samples uncovering their role in disease pathways, leading to new therapeutic targets.
This study will identify full-length circRNAs that are dysregulated in AD prefrontal-cortex samples (n = 12) compared to non-demented elderly controls (n = 12), using equal numbers of males and females and matching for age.
The protocol is derived from a Nature protocols publication. Total RNA will be extracted using a phenol-based method (SPLIT RNA extraction kit), followed by rRNA depletion. Samples will undergo RNase R treatment to degrade linear RNAs and enrich for circRNAs. Subsequently, reverse transcription will be conducted to convert RNA into cDNA strands, followed by a round of poly(A) tailing and second-strand synthesis. The Agilent Tapestation will measure the quality and concentration of cDNA. Finally, cDNA libraries will be prepared for sequencing using the ONT ligation sequencing kit v14 and sequenced on a PromethION flowcell.
Data analysis will start with processing raw sequencing data using base-calling software to generate high-quality reads. These are mapped to the human reference genome, and circRNAs quantified using CIRI-long. Importantly, we have already used publicly available ONT data to test this pipeline. Differential expression analysis will compare circRNA profiles between AD and controls, identifying dysregulated circRNAs. Bioinformatics tools like TargetScan will identify circRNA-targeted miRNAs and their sponge effect. Pathway analysis will elucidate the roles of dysregulated circRNAs in disease mechanisms. Matched omics data will be incorporated into downstream analyses.
This study will identify circular RNAs (circRNAs) that are dysregulated in the prefrontal cortex of Alzheimer’s disease (AD) patients compared to healthy controls. Using Oxford Nanopore Technology (ONT), it will generate a high-resolution dataset of full-length circRNA sequences, improving our understanding of their structure and function in AD. The research will also explore interactions between circRNAs, microRNAs and mRNAs, potentially revealing new disease mechanisms.
NIHR Exeter BRC Small Project Grant.
Dr Morteza Kouhsar, Prof Katie Lunnon, Dr Adam Smith, Dr Ehsan Pishva.
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