Quantitation and integrity evaluation of RNA genome in lentiviral vectors by direct reverse transcription-droplet digital PCR (direct RT-ddPCR)
Abstract Lentiviral vectors (LV) have proven to be powerful tools for stable gene delivery in both dividing and non-dividing cells. Approval of these LVs for use in clinical applications has been achieved by improvements in LV design. Critically important characteristics concerning quality control a...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2023-09-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-023-41644-x |
| _version_ | 1797452828170518528 |
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| author | Zhiyong He Edward J. Kwee Megan H. Cleveland Kenneth D. Cole Sheng Lin-Gibson Hua-Jun He |
| author_facet | Zhiyong He Edward J. Kwee Megan H. Cleveland Kenneth D. Cole Sheng Lin-Gibson Hua-Jun He |
| author_sort | Zhiyong He |
| collection | DOAJ |
| description | Abstract Lentiviral vectors (LV) have proven to be powerful tools for stable gene delivery in both dividing and non-dividing cells. Approval of these LVs for use in clinical applications has been achieved by improvements in LV design. Critically important characteristics concerning quality control are LV titer quantification and the detection of impurities. However, increasing evidence concerning high variability in titration assays indicates poor harmonization of the methods undertaken to date. In this study, we developed a direct reverse transcription droplet digital PCR (Direct RT-ddPCR) approach without RNA extraction and purification for estimation of LV titer and RNA genome integrity. The RNA genome integrity was assessed by RT-ddPCR assays targeted to four distant regions of the LV genome. Results of the analyses showed that direct RT-ddPCR without RNA extraction and purification performs similarly to RT-ddPCR on purified RNA from 3 different LV samples, in terms of robustness and assay variance. Interestingly, these RNA titer results were comparable to physical titers by p24 antigen ELISA (enzyme-linked immunosorbent assay). Moreover, we confirmed the partial degradation or the incomplete RNA genomes in the prepared 3 LV samples. These results may partially explain the discrepancy of the LV particle titers to functional titers. This work not only demonstrates the feasibility of direct RT-ddPCR in determining LV titers, but also provides a method that can be easily adapted for RNA integrity assessment. |
| first_indexed | 2024-03-09T15:13:21Z |
| format | Article |
| id | doaj.art-10b4b1f1c0404f13893e3a242c506026 |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-03-09T15:13:21Z |
| publishDate | 2023-09-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-10b4b1f1c0404f13893e3a242c5060262023-11-26T13:11:41ZengNature PortfolioScientific Reports2045-23222023-09-0113111110.1038/s41598-023-41644-xQuantitation and integrity evaluation of RNA genome in lentiviral vectors by direct reverse transcription-droplet digital PCR (direct RT-ddPCR)Zhiyong He0Edward J. Kwee1Megan H. Cleveland2Kenneth D. Cole3Sheng Lin-Gibson4Hua-Jun He5Material Measurement Laboratory, National Institute of Standards and TechnologyMaterial Measurement Laboratory, National Institute of Standards and TechnologyMaterial Measurement Laboratory, National Institute of Standards and TechnologyMaterial Measurement Laboratory, National Institute of Standards and TechnologyMaterial Measurement Laboratory, National Institute of Standards and TechnologyMaterial Measurement Laboratory, National Institute of Standards and TechnologyAbstract Lentiviral vectors (LV) have proven to be powerful tools for stable gene delivery in both dividing and non-dividing cells. Approval of these LVs for use in clinical applications has been achieved by improvements in LV design. Critically important characteristics concerning quality control are LV titer quantification and the detection of impurities. However, increasing evidence concerning high variability in titration assays indicates poor harmonization of the methods undertaken to date. In this study, we developed a direct reverse transcription droplet digital PCR (Direct RT-ddPCR) approach without RNA extraction and purification for estimation of LV titer and RNA genome integrity. The RNA genome integrity was assessed by RT-ddPCR assays targeted to four distant regions of the LV genome. Results of the analyses showed that direct RT-ddPCR without RNA extraction and purification performs similarly to RT-ddPCR on purified RNA from 3 different LV samples, in terms of robustness and assay variance. Interestingly, these RNA titer results were comparable to physical titers by p24 antigen ELISA (enzyme-linked immunosorbent assay). Moreover, we confirmed the partial degradation or the incomplete RNA genomes in the prepared 3 LV samples. These results may partially explain the discrepancy of the LV particle titers to functional titers. This work not only demonstrates the feasibility of direct RT-ddPCR in determining LV titers, but also provides a method that can be easily adapted for RNA integrity assessment.https://doi.org/10.1038/s41598-023-41644-x |
| spellingShingle | Zhiyong He Edward J. Kwee Megan H. Cleveland Kenneth D. Cole Sheng Lin-Gibson Hua-Jun He Quantitation and integrity evaluation of RNA genome in lentiviral vectors by direct reverse transcription-droplet digital PCR (direct RT-ddPCR) Scientific Reports |
| title | Quantitation and integrity evaluation of RNA genome in lentiviral vectors by direct reverse transcription-droplet digital PCR (direct RT-ddPCR) |
| title_full | Quantitation and integrity evaluation of RNA genome in lentiviral vectors by direct reverse transcription-droplet digital PCR (direct RT-ddPCR) |
| title_fullStr | Quantitation and integrity evaluation of RNA genome in lentiviral vectors by direct reverse transcription-droplet digital PCR (direct RT-ddPCR) |
| title_full_unstemmed | Quantitation and integrity evaluation of RNA genome in lentiviral vectors by direct reverse transcription-droplet digital PCR (direct RT-ddPCR) |
| title_short | Quantitation and integrity evaluation of RNA genome in lentiviral vectors by direct reverse transcription-droplet digital PCR (direct RT-ddPCR) |
| title_sort | quantitation and integrity evaluation of rna genome in lentiviral vectors by direct reverse transcription droplet digital pcr direct rt ddpcr |
| url | https://doi.org/10.1038/s41598-023-41644-x |
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