Computational protocol: Bovine Viral Diarrhea Virus Type 2 Impairs Macrophage Responsiveness to Toll-Like Receptor Ligation with the Exception of Toll-Like Receptor 7

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Protocol publication

[…] At 2, 6, 18, and 24 h post BVDV inoculation, cells were lysed with Buffer RLT containing 2-mercaptoethanol (Qiagen, Valencia, CA) and stored at −80°C. Total RNA was isolated using the RNeasy Mini RNA Isolation Kit (Qiagen) and genomic DNA was removed during RNA isolation using an on-column RNase-Free DNase I digestion kit (Qiagen) per manufacturer’s instructions. 500 ng of total RNA from each sample was reverse transcribed using random primers/hexamers and Superscript III (Life Technologies) to generate first strand cDNA. Primers were designed specifically for SYBR Green chemistry with the use of Primer Express v 3.0 (Applied Biosystems, Foster City, CA) or NCBI Primer Blast. Primer annealing temperature was set at 60°C with product size of 100–200 base pairs. Bovine ribosomal protein S9 (RPS9) was used as the endogenous control []. Primer set sequences designed for this study are indicated in or utilized from previous publications [, ]. An Applied Biosystems 7300 Real Time PCR Systems thermocycler was used. Amplification conditions for all genes were the same: 2 min at 50°C, 10 min at 95°C, 40 cycles of 15 s 95°C and 1 min 60°C (measure florescence step) and a dissociation step of 15 s 95°C, 1 min 60°C, 15 s 95°C, 15 s 60°C. Ct values were calculated and normalized to the endogenous control and expressed relative to medium only treatment using the 2-ΔΔCT method []. […]

Pipeline specifications

Software tools Primer Express, Primer-BLAST
Application qPCR
Organisms Bos taurus, Bovine viral diarrhea virus 2, Bovine viral diarrhea virus 1
Diseases Bacterial Infections, Hepatitis, Viral, Human, Infection, Virus Diseases, Macrophage Activation Syndrome