Computational protocol: The protozoan parasite Theileria annulata alters the differentiation state of the infected macrophage and suppresses musculoaponeurotic fibrosarcoma oncogene (MAF) transcription factors

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Protocol publication

[…] Oligonucleotides were designed for each gene using Primer3 () and Netprimer (Biosoft International) software (). The mRNA levels of each transcript were quantified by qPCR using the Platinum SYBR Green qPCR Supermix UDG kit (Invitrogen). Reactions were carried out in 20 μl vols. containing; 1× supermix (SYBR Green, Platinum taq DNA polymerase, dNTPs, uracil DNA glycosylase (UDG) and stabilizers), 0.4 μl Rox dye, 1 μl forward and reverse primers at predetermined optimal concentrations and 5 μl diluted cDNA. Amplification and detection of products was carried out using a Mx3000P PCR machine (Stratagene) with the following cycle profile: 50 °C for 2 min, 95 °C for 2 min followed by 40 cycles of 95 °C for 15 s and 60 °C for 30 s. The detection of a single product was verified by dissociation curve analysis. Each PCR experiment was carried out in triplicate and contained several non-template controls and a log10 dilution series of activated monocyte cDNA or plasmids containing the sequence of interest. The relative quantities of mRNA were calculated using the method described by . The qRT-PCR results for zinc finger protein 828 (ZNF828), previously known as Chromosome 13 open reading frame 8, were used to calculate differences in the template RNA levels and thereby standardize the results for the genes of interest. ZNF828 was previously selected from microarray and qRT-PCR analyses as a constitutively and moderately expressed gene in activated, T. annulata-infected and resting Holstein–Friesian- and Sahiwal-derived monocytes (). The relative quantity values were transformed on the log2 scale before statistical analyses to stabilize the variance. The effects of cell differentiation, infection and buparvaquone treatment were examined by t-test analysis (Genstat 10.2, Lawes Agricultural Trust, Rothamsted). […]

Pipeline specifications

Software tools Primer3, NetPrimer
Application qPCR
Organisms Theileria annulata, Bos taurus, Trilophidia annulata
Diseases Fibrosarcoma, Theileriasis, Tick-Borne Diseases