Stratagene mx3005p system

DNA was extracted from whole blood using MagVet universal isolation kit (Life Technologies) at different days throughout the study. Samples were assayed in duplicate for the presence of viral DNA by quantitative PCR (qPCR) on a Stratagene Mx3005P system (Agilent Technologies, Santa Clara, CA, USA) following a protocol modified (44 (link)) from using the primers Vp72 sense (CTG CTC ATG GTA TCA ATC TTA TCG A) and Vp72 antisense (GAT ACC ACA AGA TC[AG] GCC GT) and the probe 5′-(6-carboxyfluorescein [FAM])-CCA CGG GAG GAA TAC CAA CCC AGT G-3′-(6-carboxytetramethylrhodamine [TAMRA]) (44 (link)). A standard curve was prepared from a p72 mimic plasmid by making serial dilution ranging from 10⁸ to 10¹ copies/mL. Results were reported as log₁₀ genome copies/mL.

The mouse lung total RNA utilized for microarrays was also used for qPCR experiments. For reverse transcription, a ThermoScript RT-PCR system with random hexamer primers was used according to the manufacturer instructions (Life Technologies, Grand Island, NY). Briefly, 200 ng of total lung RNA from each mouse was used for cDNA synthesis. Control reactions without reverse transcriptase were also performed to exclude significant DNA contamination. The amplified cDNA samples were diluted 50-fold and used for qPCR with a SYBR green qPCR super Mix Universal (Life Technologies, Grand Island, NY) in a Stratagene Mx3005p system (Agilent Technologies, Santa Clara, CA). The 10 μL PCR mixture contained 1.5 μL (5 ng) of cDNA template, 5 μL of SYBR Green Master Mix, 2 μL of primers (10 pM), and 1.5 μL nuclease-free water. The PCR conditions were one cycle of 10 min at 94°C followed by 40 cycles of amplification, each with 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s. The oligonucleotide primers used for qPCR are listed in Supplementary Table  1 in Supplementary Material available online at http://dx.doi.org/10.1155/2015/385402. Threshold cycle (Ct) values of test and control (Gapdh) genes were measured and used to calculate the fold change in expression using Mx4000 software (Agilent Technologies, Santa Clara, CA).

qPCR was performed on a Stratagene Mx3005P system (Agilent Technologies) using UltraSYBR mixture (CWBIO) according to the manufacturer's instructions (10 µl of 2 × UltraSYBR mixture, 0.2 µM of forward primer, 0.2 µM of reverse primer, and 10 ng of cDNA in a total volume of 20 µl). The qPCR thermal profile included an initial denaturation at 95°C for 10 min followed by quantification for 45 cycles (95°C for 10 s, annealing at 60°C for 30 s, and extension at 72°C for 32 s). Next, the dissolution curve was analyzed (95°C for 15 s, 60°C for 1 min, 95°C for 15 s, 60°C for 15 s). The relative change in gene expression was analyzed using 2‐^ΔΔCT method. β‐actin was used as the reference gene. Primer sequences are listed in Table 1.