ArticleScientific reports2023
A duplex droplet digital PCR assay for absolute quantification and characterization of long self-amplifying RNA.
Article in Scientific reports, 2023. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 2 papers.
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Who cites it
2 citing papers in PubMed, 4 citations in OpenAlex.
- Transgene sequence codon optimization and composition determines replication competence of self-amplifying RNA.Molecular therapy : the journal of the American Society of Gene Therapy · 2026Article
- Critical Evaluation of Key Methods for RNA Quantification.International journal of molecular sciences · 2026Review
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Authors and funding
3 authors at 1 institution in 1 country.
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Abstract
Self-amplifying messenger ribonucleic acid (saRNA) provides extended expression of genes of interest by encoding an alphavirus-derived RNA replicase and thus is 2-3 times larger than conventional messenger RNA. However, quality assessment of long RNA transcripts is challenging using standard techniques. Here, we utilized a multiplex droplet digital polymerase chain reaction (ddPCR) assay to assess the quality of saRNA produced from an in vitro transcription reaction and the replication kinetics in human cell lines. Using the one-step reverse transcription ddPCR, we show that an in vitro transcription generates 50-60% full-length saRNA transcripts. However, we note that the two-step reverse transcription ddPCR assay results in a 20% decrease from results obtained using the one-step and confirmed using capillary gel electrophoresis. Additionally, we provided three formulas that differ in the level of stringency and assumptions made to calculate the fraction of intact saRNA. Using ddPCR, we also showed that subgenomic transcripts of saRNA were 19-to-108-fold higher than genomic transcripts at different hours post-transfection of mammalian cells in copies. Therefore, we demonstrate that multiplex ddPCR is well suited for quality assessment of long RNA and replication kinetics of saRNA based on absolute quantification.
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