Rotor gene 6000

Total RNA was extracted from ∼6 × 10⁵ uninfected or infected BHK-21 cells (5 PFU/cell, 8 h) with TRIzol (Invitrogen). Genomic DNA was eliminated by treatment with 2 U of DNase I (Turbo DNase; Ambion). DNase-treated RNA (500 ng) was retrotranscribed using 2.5 μM oligo(dT) primer and 10 U/μl Superscript III reverse transcriptase (Invitrogen) in a 20-μl reaction volume. All samples were treated with 2 U RNase H (Invitrogen), and 2 μl of a 1/10 dilution was used in each quantitative PCR (qPCR; Dynamo Flash SYBR green qPCR kit). Primer pairs, designed with Primer3, were M11_F1 (5′-ACCCAGGAGTTTAGAAGGCA) and M11_ R1 (5′-CAACGAGGTGAAAAGTTTGGAC-3′) for M11, M3_F1 (5′-AACATCAAGCTGACCCCAAC-3′) and M3_R1 (5′-GGGTGTGGACTTCAACTTCC-3′) for M3, ORF63_F1 (5′-GCGCTGACAACGACTCTATT-3′) and ORF63_R1 (5′-ATTTGGGCAGGTGGGGTATA-3′) for ORF63, and GAPDH_hams_F2 (5′-ACCTGCCAAGTATGAGGACA-3′) and GAPDH_hams_R2 (5′-AAGGTGGAAGAGTGGGAGTC-3′) for hamster GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Reactions were performed in triplicate using a Rotor Gene 6000 and analyzed with Rotor Gene 6000 software (Corbett Life Science). ΔΔC_T values (where C_T is threshold cycle) were calculated. Samples prepared in parallel without addition of reverse transcriptase confirmed that contamination with genomic DNA was negligible (data not shown).

cDNA was synthesized from total RNA using the QuantiTech Reverse Transcription Kit (QIAGEN). SensiFast SYBR No-ROX Kit (Bioline) was used for qPCR with a Corbett Rotor-Gene 6000 machine driven by Rotor-Gene 6000 v1.7 software. The primers were designed using Primer3 online software (http://bioinfo.ut.ee/primer3-0.4.0/primer3/). At least one primer of each primer pair aligned to an exon–exon junction. Primers designed for ama-1 and act-1 genes were used for normalization. The efficiency of the primer pairs was tested by setting a standard curve using a serial dilution of cDNA, and the specificity of the primers was monitored by analyzing the melting curves of each reaction. Data from triplicate reactions were analyzed using a 2^−ΔΔCt method. Biological replicates were used to confirm the results. For statistical analysis, we used the Student’s t-test to compare the relative mRNA level of different genetic variants.

Leukocyte DNA was extracted by the salting-out method [26] (link). Telomere length was measured using a validated quantitative PCR-based method as previously described [27] (link). Briefly, the relative telomere length was calculated as the ratio of telomere repeats to single-copy gene (SCG) copies (T/S ratio). For each sample the quantity of telomere repeats and the quantity of SCG copies were determined in comparison to a reference sample in a telomere and a SCG quantitative PCR, respectively. The raw data from each PCR was analysed using the comparative quantification analysis (Rotor-Gene 6000 software, Corbett Research Ltd., Cambridge, UK). All PCRs were performed on the Rotor-Gene 6000 (Corbett Research Ltd., Cambridge, UK). The coefficient of variation in repeated measurements was 5.6%.