A rapid and efficient branched DNA hybridization assay to titer lentiviral vectors.
A robust assay to titer lentiviral vectors is imperative to qualifying their use in drug discovery, target validation and clinical applications. In this study, a novel branched DNA based hybridization assay was developed to titer lentiviral vectors by quantifying viral RNA genome copy numbers from viral lysates without having to purify viral RNA, and this approach was compared with other non-functional (p24 protein ELISA and viral RT-qPCR) and a functional method (reporter gene expression) used commonly. The RT-qPCR method requires purification of viral RNA and the accuracy of titration therefore depends on the efficiency of purification; this requirement is ameliorated in the hybridization assay as RNA is measured directly in viral lysates. The present study indicates that the hybridization based titration assay performed on viral lysates was more accurate and has additional advantages of being rapid, robust and not dependent on transduction efficiency in different cell types.
Nair
Ayyappan
A
Bayer Health Care, 2600 Hilltop Drive, P.O. Box 4099, Richmond, CA 94804, United States. ayyapan.nair@bayer.com
Xie
Jinger
J
Joshi
Sarasijam
S
Harden
Paul
P
Davies
Joan
J
Hermiston
Terry
T
D021121:Branched DNA Signal Amplification Assay / Q000379:methods*; D002460:Cell Line; D018628:Gene Dosage; D005822:Genetic Vectors / Q000235:genetics*; D016679:Genome, Viral; D006367:HeLa Cells; D006801:Humans; D016086:Lentivirus / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology; D012367:RNA, Viral / Q000032:analysis / Q000235:genetics; D013601:T-Lymphocytes; D013997:Time Factors
D012367:RNA, Viral