Recommendations for detection, validation, and evaluation of RNA editing events in cardiovascular and neurological/neurodegenerative diseases

Figures

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  Figure 1. Experimental pipeline for RNA editing studies (A) Summary of the process of sample collection, storage, and RNA isolation, depending on the type of sample. (B) The required steps for RNA-seq library preparation, and the existing RNA-seq approaches depending on the average size and type of RNA. (C) Summary of the main existing bioinformatic tools for RNA editing analysis. (D) Summary of the workflow of utilizing software tools for the identification of potential biological function. (E) Summary of the workflow for experimental validation of RNA editing events, using different PCR techniques. (F) Summary of various experimental methods for the detection of functional effects of RNA editing events, and confirmation of clinical relevance of RNA editing in patient samples. Created with BioRender.com.

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  Figure 2. Steps for plasma handling and processing Several crucial steps and pitfalls are highlighted to both warrant high-quality RNA yields from plasma and circulating extracellular vesicles and to avoid hemolysis and platelet contamination. Created with BioRender.com.

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  Figure 3. Overview of the main approaches for small RNA-seq analysis (A) Original two-adaptor ligation protocol for small RNA-seq analysis. (B) Improved two-adaptor ligation methods for small RNA-seq analysis. The reduction of PCR or ligation bias can be accomplished through randomized adaptors in the ligation steps or unique molecular identifiers in the amplification step. (C) Ligation-free approach for small RNA-seq analysis through polyadenylation of the 3′ end and template switching method. Created with BioRender.com.

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  Figure 4. Schematic illustration for JACUSA2 workflow analysis JACUSA2 tool predicts single-nucleotide differences from both RNA-DNA (RDDs) and RNA-RNA (RRDs) sequencing data. PCR duplicates are being marked and variants in the start and end of the genome, in INDELs, in intronic regions and in homopolymers are being filtered accordingly to prevent false-positive RNA editing events. Created with BioRender.com.

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  Figure 5. Schematic illustration for SPRINT bioinformatics analysis SPRINT enables the detection of A-to-I and C-to-U editing sites by mapping reads to the corresponding reference genome using Burrows-Wheeler aligner and clustering RES-based and SNP-based SNV duplets. Both regular RES from mapped reads and hyper RES from recovered remapped reads can be identified. Created with BioRender.com.

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  Figure 6. Experimental validation and clinical relevance of RNA editing events (A) Various methods to discover, detect, validate, and quantify individual RNA editing sites. Top to bottom, the panel shows RNA-seq, allele-specific RT-qPCR, two-tailed RT-qPCR, mmPCR sequencing, and restriction fragment-length polymorphism analysis of cDNA by southern blotting. (B) RT-qPCR to help in studying the impact of RNA editing on the expression of parent RNAs (mRNAs, lncRNAs, circRNAs, or miRNAs) or their downstream targets. Western blotting helps to study the effect of RNA editing on the protein expression of edited parent coding RNA or its downstream protein target/s. Luciferase reporter gene assays help in validate any alterations in the targetome of edited RNAs. (C) Methods to study the role of RNA editing enzymes. From top to bottom the panel shows RT-qPCR, western blotting, RNA binding protein immunoprecipitation (RIP), and various methods for DNA/RNA modulation. (D) Methods to study the functional effect of RNA editing events. CRISPR-based techniques such as RESTORE and RESCUE can be used to generate unedited and edited RNA molecules for in vitro or in vivo studies. Phenotypic assays such as…