Real-time quantitative PCR analysis of viral transcription.
Whole-genome profiling using DNA arrays has led to tremendous advances in our understanding of cell biology. It has had similar success when applied to large viral genomes, such as the herpesviruses. Unfortunately, most DNA arrays still require specialized and expensive resources and, generally, large amounts of input RNA. An alternative approach is to query entire viral genomes using real-time quantitative PCR. We have designed such PCR-based arrays for every open reading frame of human herpesvirus 8 and describe here the general design criteria, validation procedures, and detailed application to quantify viral mRNAs. This should provide a useful resource either for whole-genome arrays or just to measure transcription of any one particular mRNA of interest. Because these arrays are RT-PCR-based, they are inherently more sensitive and robust than current hybridization-based approaches and are ideally suited to query viral gene expression in models of pathogenesis.
Papin
James
J
Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA.
Vahrson
Wolfgang
W
Hines-Boykin
Rebecca
R
Dittmer
Dirk P
DP
D001483:Base Sequence; D017931:DNA Primers; D004279:DNA, Viral / Q000378:metabolism*; D004663:Encephalitis Virus, Eastern Equine / Q000235:genetics / Q000378:metabolism; D006564:Herpesviridae / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012984:Software; D014158:Transcription, Genetic / Q000502:physiology*
D017931:DNA Primers; D004279:DNA, Viral