Computational protocol: Gene Expression in the Skin of Dogs Sensitized to the House Dust Mite Dermatophagoides farinae

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Protocol publication

[…] The results of the MA analysis were verified by validating the expression of selected DEG by quantitative real-time RT-PCR (qPCR). Specific primers were designed using the NCBI Primer-BLAST tool (http://www.ncbi.nlm.nih.gov/tools/primer-blast/) and synthesized by Thermo Fisher Scientific GmbH (http://www.thermoscientific.com/biopolymers; Germany). The sequences of the used qRT-PCR-Primer are shown in Table S2. The same RNA samples were used for qPCR and MA analysis. First-strand cDNA was synthesized starting from 1 µg of total RNA with the Sprint RT Complete-Double PrePrimed 48-well strips Kit (Takara Bio Europe/Clontech, Saint-Germain-en-Laye, France) according to the manufacturer’s instructions. A two-step qPCR experiment was performed (PMID 22031715) (). The Power SYBR Green PCR Master Mix and RT-PCR (Applied Biosystems, Darmstadt, Germany) was applied in the thermal cycler (StepOne Real-Time PCR System; Applied Biosystems) and evaluated with the qPCR StepOne Software (V 2.2.2; Applied Biosystems. The cycle thresholds (CTs) determined for the target genes were normalized against the geometric mean of the reference genes RLPL13A, LOC479750 (CCZ1) as described by and UBB to obtain ∆CT values. Quantitative PCR results were statistically evaluated with the same parameters as microarray data in limma. […]

Pipeline specifications

Software tools Primer-BLAST, limma
Application qPCR
Organisms Canis lupus familiaris, Dermatophagoides farinae, Homo sapiens
Diseases Dermatitis, Atopic, Immune System Diseases, Skin Diseases