Computational protocol: Design and evaluation of a unique SYBR Green real-time RT-PCR assay for quantification of five major cytokines in cattle, sheep and goats

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Protocol publication

[…] The RT-qPCR method was checked by amplification, in duplicate, of cDNA synthesized from (i) Tempus™ total RNA from three different animals (absolute quantification), and (ii) PBMC (ConcanavalinA-stimulated and unstimulated) total RNA from three independent experiments (relative expression).Absolute quantification results are expressed as the mean number of copies (nc) from six amplification values with standard deviation. Nc was calculated using the following equation: Nc=(Relative amount of target (in ng) × 10−9)/(DNA length(dp) x 650) × 6.022 × 1023. The relative amount of target (in ng) was calculated using the calibration curve and the following equation: Relative amount of target (in ng)=10 ((number of Cq-intercept)/slope).Relative expression was calculated with efficiency correction using the relative expression software tool (REST) []. Multiple reference gene normalization was chosen.Gene expression stability of the five candidate reference genes was determined in PBMC samples and calculated using the geNorm application in Microsoft Excel []. Stability values (M-value) and pairwise variations (V-score) were calculated and the optimal number of reference genes required for relative expression was determined. In addition, the best stable combination of reference genes was selected with NormFinder Excel Add-In []. The relative expression ratio (Er) of cytokine genes in ConcanavalinA-stimulated cells (n=3) compared to unstimulated cells (n=3) was calculated using the quantification of cytokine and reference gene as number of copies. Expression variation for each cytokine gene was provided by REST []. […]

Pipeline specifications

Software tools REST, NormFinder
Application qPCR
Organisms Ovis aries, Capra hircus, Bos taurus
Diseases Animal Diseases