The DOI matches its Crossref record; 13 of the 13 references it cites were found in a registry or an index.
Strand-specific real-time RT-PCR for distinguishing influenza vRNA, cRNA, and mRNA
Open accessAbstract
Real-time RT-PCR is used to quantify individual influenza viral RNAs. However, conventional real-time RT-PCR, using strand-specific primers, has been shown to produce not only the anticipated strand-specific products, but also substantial amounts of non-strand-specific products, indicating lack of specificity. Therefore, in this study, a novel strand-specific real-time RT-PCR method was established to quantify the three types of influenza viral RNA (vRNA, cRNA, and mRNA) separately. This method is based on reverse transcription using tagged primers to add a tag sequence at the 5 end and the hot-start method. Real-time PCR using the tag portion as the forward primer and a segment-specific reverse primer ensured the specificity for quantifying the three types of RNA. Using this method, specific target RNA was detected at 100 – 100,000 folds higher level than other types of RNA. This method was also used to evaluate the vRNA, cRNA, and mRNA levels of segments 5 and 6 in MDCK cells infected with influenza A virus at different time points post-infection. The cRNA level was 1/10 to 1/100 lower than that of the vRNA and mRNA. Moreover, different dynamics of vRNA, cRNA, and mRNA synthesis were observed; the copy number of the vRNA gradually increased throughout infection, the cRNA increased and then plateaued, while the mRNA increased and then decreased. This novel method thus provides data critical for understanding the influenza virus life cycle, including transcription, replication, and genome incorporation into virions.
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Generation of influenza A viruses entirely from cloned cDNAs
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Self-citationThe biology of influenza viruses
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Genome-wide RNAi screen identifies human host factors crucial for influenza virus replication
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NS2/NEP protein regulates transcription and replication of the influenza virus RNA genome
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Cited by
Species difference in ANP32A underlies influenza A virus polymerase host restriction
Long, J. S., Giotis, E. S., Moncorgé, O., Frise, R., Mistry, B., James, J., Morisson, M., Iqbal, M., Vignal, A., Skinner, M. A., et al. (2016) · Nature · doi:10.1038/nature16474 · Cited by 323
Influenza Virus-Host Interactome Screen as a Platform for Antiviral Drug Development
Watanabe, T., Kawakami, E., Shoemaker, J. E., Lopes, T. J. S., Matsuoka, Y., Tomita, Y., Kozuka-Hata, H., Gorai, T., Kuwahara, T., Takeda, E., et al. (2014) · Cell Host & Microbe · doi:10.1016/j.chom.2014.11.002 · Cited by 290
Self-citationBroadbent, A. J., Santos, C. P., Anafu, A., Wimmer, E., Mueller, S., & Subbarao, K. (2016) · Vaccine · doi:10.1016/j.vaccine.2015.11.054 · Cited by 79
Metadata
- Authors
- Kawakami, E., Watanabe, T., Fujii, K., Goto, H., Watanabe, S., Noda, T., & Kawaoka, Y.
- Venue
- Journal of Virological Methods
- Publisher
- Elsevier BV
- Published
- 2011-04
- Volume
- 173
- Issue
- 1
- Pages
- 1-6
- ISSN
- 0166-0934 (ISSN-L)
- OpenAlex
- W2099533162
- Cited by
- 288
- References
- 13
- FWCI
- 4.82
- Type
- journal article
- Language
- en
- Open access
- green
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- The paper
- Kawakami, E., Watanabe, T., Fujii, K., Goto, H., Watanabe, S., Noda, T., & Kawaoka, Y. (2011). Strand-specific real-time RT-PCR for distinguishing influenza vRNA, cRNA, and mRNA. Journal of Virological Methods. https://doi.org/10.1016/j.jviromet.2010.12.014
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Contains information from the Semantic Scholar Academic Graph Datasets, made available under the ODC Attribution License (ODC-BY 1.0).
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