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.
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.
Li
Yi Ping
YP
Department of Poultry, Fish and Fur Animals, Danish Institute for Food and Veterinary Research, DK-8200 Aarhus, Denmark.
Bang
Dang D
DD
Handberg
Kurt J
KJ
Jorgensen
Poul H
PH
Zhang
Man Fu
MF
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*
D012333:RNA, Messenger