title: Evaluation of the suitability of six host genes as internal control in real-time RT-PCR assays in chicken embryo cell cultures infected with infectious bursal disease virus.
abstract: Infectious bursal disease virus (IBDV) can cause disease in chickens characterized by immunosuppression and high mortality. Currently, real-time RT-PCR has been used to quantitate virus-specific RNA and to better understand host response to infection. However, normalization of quantitative real-time RT-PCR is needed to a suitable internal control. We thus investigated the expression pattern of six chicken genes, including beta-actin, 28S rRNA, 18S rRNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), TATA box-binding protein (TBP) and beta-2-microglobulin, in chicken embryo (CE) cell cultures following a 7-day IBDV infection. The CE cells were inoculated with various multiplicity of infection (MOI) of IBDV vaccine strain Bursine-2, the expression of genes was measured by quantitative real-time PCR-based on cDNA synthesized from either normalized (100 ng) or non-normalized (10 microl) total RNA. The results showed that beta-actin, 28S rRNA, 18S rRNA and GAPDH were the most constantly expressed genes, while TBP and beta-2-microglobulin were markedly induced during the infection course. Of these constant expressed genes, 28S rRNA and 18S rRNA are highly expressed; beta-actin intermediately expressed and GAPDH had a lower expression level in CE cell cultures. Also, beta-actin showed no significant variation in both normalized and non-normalized assays and virus dose-independent of inoculation, while other genes did. beta-Actin was further successfully used as an internal control to quantitate Bursine-2 virus-specific RNA load in CE cell cultures. Thus, beta-actin was suggested as a suitable internal control in studying gene expression as well as virus-specific RNA load in CE cell after IBDV infection.
authors.affiliation: Department of Poultry, Fish and Fur Animals, Danish Institute for Food and Veterinary Research, DK-8200 Aarhus, Denmark.
authors.forename: Yi Ping
authors.initials: YP
authors.lastname: Li
authors.forename: Dang D
authors.initials: DD
authors.lastname: Bang
authors.forename: Kurt J
authors.initials: KJ
authors.lastname: Handberg
authors.forename: Poul H
authors.initials: PH
authors.lastname: Jorgensen
authors.forename: Man Fu
authors.initials: MF
authors.lastname: Zhang
mesh_terms: D000818:Animals; D018175:Birnaviridae Infections / Q000235:genetics* / Q000382:microbiology; D002470:Cell Survival; D002478:Cells, Cultured; D002642:Chick Embryo; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000235:genetics; D007243:Infectious bursal disease virus / Q000235:genetics* / Q000502:physiology*; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards*
chemical_list: D012333:RNA, Messenger