A duplex droplet digital PCR assay for absolute quantification and characterization of long self-amplifying RNA.

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 conf

SUBMITTER: Casmil IC 

PROVIDER: S-EPMC10624827 | biostudies-literature | 2023 Nov

REPOSITORIES: biostudies-literature