Real time PCR for monitoring regulation of host gene expression in herpes simplex virus type 1-infected human diploid cells.
Herpes simplex virus type 1 (HSV-1) induces prominent shifts in the rates of transcription of host cellular genes of relevance for the outcome of the viral infection. The quantitative analysis of transcription may be obscured by virus-induced alterations in the levels of RNA encoded by cellular housekeeping genes that are used commonly for normalisation of real time reverse transcription PCR (RT-qPCR). In the present study, we analysed beta-actin, GAPDH and 18S rRNA for their usefulness in normalisation of RT-qPCR analysis of the transcription of the HSV-1 gamma gB-1 gene and FUT5, a cellular gene induced by viral infection. The transcription of these genes was monitored in a TaqMan-based real time RT-PCR system over a 24h interval of virus infection of human embryonic lung fibroblasts. The levels of gB-1 and FUT5 RNA were normalised via difference in the threshold cycle (deltaC(t)) values relative to each and one of the housekeeping genes or calculated in relation to the number of infected cells without any further normalisation. The levels of RNA encoded by beta-actin or GAPDH were found to vary by several orders of magnitude during HSV-1 infection, introducing large errors in the estimation of the gB-1 and FUT5 RNA levels. In contrast, the variation of C(t) values for 18S rRNA was less than one cycle during 24h period of HSV-1 infection. The FUT5 and gB-1 RNA figures obtained by DeltaC(t) normalisation relative 18S rRNA were identical to those calculated in relation to the number of infected cells. These data recommend 18S rRNA for normalisation in HSV-1-infected human cells but discourage the use of beta-actin and GAPDH RNA for this purpose. By applying these procedures, it was shown that the transcription of FUT5 was increased by 50-fold 5-24h after HSV-1 infection and 200-fold by the inhibition of viral DNA replication in HSV-infected cells.
Nyström
Kristina
K
Clinical Virology, University of Göteborg, Guldhedsgatan 10B, S-413 46 Göteborg, Sweden.
Biller
Marlene
M
Grahn
Ammi
A
Lindh
Magnus
M
Larson
Göran
G
Olofsson
Sigvard
S
D000199:Actins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D004171:Diploidy; D005347:Fibroblasts / Q000821:virology*; D005647:Fucosyltransferases / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D006561:Herpes Simplex / Q000821:virology; D018259:Herpesvirus 1, Human / Q000235:genetics / Q000378:metabolism / Q000472:pathogenicity*; D006801:Humans; D012337:RNA, Ribosomal, 18S / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D019914:Taq Polymerase / Q000378:metabolism; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism
D000199:Actins; D012337:RNA, Ribosomal, 18S; D014759:Viral Envelope Proteins; C040495:glycoprotein B, Simplexvirus; D005647:Fucosyltransferases; D019914:Taq Polymerase