Figure 2. Detection methods for RNA... | American Society of Hematology

Figure 2.

$Detection methods for RNA editing events. (A) Sanger sequencing; detect the RNA editing levels by measuring ratios of G/(G+A) or T/(T+C) peaks. (B) Bulk-RNA sequencing; used to identify differences between RNA and DNA sequences, aided by REDItools suite. (C) mmPCR sequencing; uniformly and simultaneously amplify up to 960 loci in 48 samples, followed by sequencing. (D) Single-cell RNA sequencing; after preprocessing, alignment, UMI/barcode aggregation, and filtering steps, RNA editing sites can be annotated using tools such as ANNOVAR. (E) Digital PCR is a highly sensitive technique that allows for the detection of rare RNA editing events in hot spot regions by partitioning thousands of PCR reactions and using ≥2 fluorescent probes to determine the absolute proportions of sequence variants. (F) RESS-qPCR; detects wild-type (A) or edited (I) bases by incorporating mismatches in primers. Primer design strategy (i) uses the tetra-primer amplification refractory mutation system (ARMS) principles; strategy (ii) is used for positions that are incompatible with the Tetra-primer ARMS method due to significant differences in GC content upstream and downstream of the edited position. (G) Two-tailed qPCR for isomiRs. Specialized hemiprobes are used as primers for reverse transcription of both edited and unedited miRNAs. (H) LNA primer for isomiRs. This approach leverages tailored qPCR primers to detect miRNA isoforms with different melting temperatures, ensuring high sensitivity and accuracy in distinguishing miRNA variations. cDNA, complementary DNA; FW, forward; isomiRs, isoform of miRNAs; LNA, locked nucleic acid; mmPCR, microfluidics-based multiplex PCR; nt, nucleotides; pos, positive; RESS-qPCR, RNA editing site-specifc quantitative PCR; Rev, reverse; UMI, unique molecular identifier; wt, wide type.$

Detection methods for RNA editing events. (A) Sanger sequencing; detect the RNA editing levels by measuring ratios of G/(G+A) or T/(T+C) peaks. (B) Bulk-RNA sequencing; used to identify differences between RNA and DNA sequences, aided by REDItools suite. (C) mmPCR sequencing; uniformly and simultaneously amplify up to 960 loci in 48 samples, followed by sequencing. (D) Single-cell RNA sequencing; after preprocessing, alignment, UMI/barcode aggregation, and filtering steps, RNA editing sites can be annotated using tools such as ANNOVAR. (E) Digital PCR is a highly sensitive technique that allows for the detection of rare RNA editing events in hot spot regions by partitioning thousands of PCR reactions and using ≥2 fluorescent probes to determine the absolute proportions of sequence variants. (F) RESS-qPCR; detects wild-type (A) or edited (I) bases by incorporating mismatches in primers. Primer design strategy (i) uses the tetra-primer amplification refractory mutation system (ARMS) principles; strategy (ii) is used for positions that are incompatible with the Tetra-primer ARMS method due to significant differences in GC content upstream and downstream of the edited position. (G) Two-tailed qPCR for isomiRs. Specialized hemiprobes are used as primers for reverse transcription of both edited and unedited miRNAs. (H) LNA primer for isomiRs. This approach leverages tailored qPCR primers to detect miRNA isoforms with different melting temperatures, ensuring high sensitivity and accuracy in distinguishing miRNA variations. cDNA, complementary DNA; FW, forward; isomiRs, isoform of miRNAs; LNA, locked nucleic acid; mmPCR, microfluidics-based multiplex PCR; nt, nucleotides; pos, positive; RESS-qPCR, RNA editing site-specifc quantitative PCR; Rev, reverse; UMI, unique molecular identifier; wt, wide type.

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