دورية أكاديمية
A duplex droplet digital PCR assay for absolute quantification and characterization of long self-amplifying RNA
| العنوان: | A duplex droplet digital PCR assay for absolute quantification and characterization of long self-amplifying RNA |
|---|---|
| المؤلفون: | Irafasha C. Casmil, Cynthia Huang, Anna K. Blakney |
| المصدر: | Scientific Reports, Vol 13, Iss 1, Pp 1-12 (2023) |
| بيانات النشر: | Nature Portfolio, 2023. |
| سنة النشر: | 2023 |
| المجموعة: | LCC:Medicine LCC:Science |
| مصطلحات موضوعية: | Medicine, Science |
| الوصف: | 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. |
| نوع الوثيقة: | article |
| وصف الملف: | electronic resource |
| اللغة: | English |
| تدمد: | 2045-2322 |
| Relation: | https://doaj.org/toc/2045-2322 |
| DOI: | 10.1038/s41598-023-46314-6 |
| URL الوصول: | https://doaj.org/article/f0197f1cfe464e06845870c0304898c4 |
| رقم الأكسشن: | edsdoj.f0197f1cfe464e06845870c0304898c4 |
| قاعدة البيانات: | Directory of Open Access Journals |
Find this article in full text from Springer Verlag. 
Full Text Finder | تدمد: | 20452322 |
|---|---|
| DOI: | 10.1038/s41598-023-46314-6 |