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Quantification of infectious recombinant murine MSCV-based retroviruses using PCR approach


Nik Abd Rahman, Nik Mohd Afizan and Allaudin, Zeenathul Nazariah and Ismail, Ruzila and Mustafa, Nor Hidayah and Mohd Lila, Mohd Azmi (2010) Quantification of infectious recombinant murine MSCV-based retroviruses using PCR approach. Research Journal of International Studies, 16. pp. 5-14. ISSN 1453-212X


The current available molecular method to detect the retroviral-mediated gene expression is reverse transcriptase-polymerase chain reaction (RT-PCR). However, the conventional PCR is time consuming, prone to error and not sensitive. In this paper, we performed a real-time PCR assay to quantify retroviral mediated gene expression in the supernatant of infectious recombinant virus. We documented an optimized methodology for accurate and reproducible quantification of retrovirus-based gene expression which enables rapid and quantitative determination of sample gene expression. We have also compared the performance of the assay with our routine RT-PCR assay. RNA was extracted from infectious recombinant retrovirus at 48 h post-infection and assayed for VP3 gene. The standard curve from linear DNA standards showed high sensitivity and good linearity (R2 = 0.9902 for VP3 standards graphs), ranged from 102 to 108 copies of comparable accuracy to current quantification real-time PCR methods. The present study presumes a copy number of transgene expression to equal a molecule of infectious recombinant virus particle. Another advantage, this concept could determine vector stability by comparison of infectious particles, total particles and particles with RNA. In comparison to gel-based RTPCR, we conclude that real-time PCR as a better approach in gene expression quantification.

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Additional Metadata

Item Type: Article
Divisions: Faculty of Veterinary Medicine
Publisher: EuroJournals Publishing
Keywords: Infectious; Retrovirus; Real-time PCR
Depositing User: Azizan Arshad
Date Deposited: 19 Nov 2013 08:24
Last Modified: 30 Oct 2019 06:16
URI: http://psasir.upm.edu.my/id/eprint/16410
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