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Epstein–Barr virus-encoded LMP1 induces a hyperproliferative and inflammatory gene expression programme in cultured keratinocytes, by M. A. Morris, C. W. Dawson, W. Wei, J. D. O'Neil, S. E. Stewart, J. Jia, A. I. Bell, L. S. Young and J. R. Arrand

Journal of General Virology vol. 89, part 11, pp. 2806 - 2820

Supplementary Methods

ExpressArt RNA amplification. Briefly, 5 ng total RNA was used as template in a cDNA synthesis reaction using oligo(dT) as primer. Double-stranded cDNA was generated using 'Box-trinucleotide' primers that prime preferentially near the 3′ ends of the first strand. The double-stranded cDNA was denatured and used as template for synthesis of cDNA, this time using a T7/oligo (dT) primer. The resulting double-stranded cDNA was the template for first-round amplification via RNA synthesis using T7 RNA polymerase, yielding RNA with defined sequences at each end (T7 promoter and 'box-trinucleotide').
In the second round, amplified RNA from the first round was reverse-transcribed to cDNA using 'box-trinucleotide' as primer and then converted to double-stranded cDNA by priming with T7/oligo(dT). This cDNA was then subjected to a third-round amplification similar to the second. The final IVT step introduces the biotinylated nucleotides.

Microfluidics card Q-PCR. One-step RT-PCRs were performed using a QuantiTect Probe RT-PCR kit (Qiagen). For each fill reservoir on the card, the reaction mixture contained 50 µl RT-PCR master mix, 1 µl QuantiTect RT mix and 100 ng total RNA in a final volume of 100 µl. Reaction conditions were 50 °C for 30 min, 95 °C for 15 min followed by 40 cycles of 96 °C for 30 s, 59.7 °C for 1 min.

Individual Q-PCRs. Individual Q-PCRs were performed in 96-well optical reaction plates on an Applied Biosystems 7900HT Fast Real-Time PCR System, using β2-microglobulin as standard. Reactions contained 5 µl cDNA, TaqMan Universal PCR Master Mix and Assays-on-Demand Gene Expression Assays, according to the manufacturer's instructions.

Cytokine array and ELISA. The secretory profile of 42 different cytokines in the culture supernatant of uninduced and induced cells was examined using the antibody-based RayBio human cytokine array V (RayBiotech Inc.) according to the manufacturer's protocol. ELISA was carried out according to the manufacturer's instructions (Pelikine) and is described elsewhere (Eliopoulos et al., 1997).

Reference

Eliopoulos, A. G., Stack, M., Dawson, C. W., Kaye, K. M., Hodgkin, L., Sihota, S., Rowe, M. & Young, L. S. (1997). Epstein-Barr virus-encoded LMP1 and CD40 mediate IL-6 production in epithelial cells via an NF-kappaB pathway involving TNF receptor-associated factors. Oncogene 14, 2899–2916. Medline

Supplementary Table S1. Antibodies used in this study

Supplementary Table S2. Applied Biosystems Q-PCR assays used in this work

Supplementary Table S3. RT-PCR primers used in this work

Supplementary Table S4. Individual results for differentially expressed genes as determined by RP, SAM and dChip and by Q-PCR

[PDF file of Supplementary Tables S1-S4] (159 KB)

Supplementary Fig. S1. Labelling and amplification protocols used in this study. [PDF] (16 KB)

This Article Abstract Full Text Services Email this article to a friend Alert me to new issues of the journal Citing Articles Citing Articles via CrossRef