Skip to main content

Table 3 Assay performances for five cytokine genes for bovine, caprine and ovine gene expression

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

Target gene Species Tm (°C) Intercept Slope E (%) Inter-assay variability LOD nc
Mean Cq SD Cq Mean nc SD nc
IL-4 cattle 83.9 2.02 −3.372 102 28.5 0.2 25 3 9
goat 1.37 −3.276 99 28.6 0.2 30 4 11
sheep 1.39 −3.283 99 28.1 0.1 38 2 5
IL-10 cattle 86.6 0.27 −3.50 95 27.6 0.2 104 16 6
goat 0.91 −3.33 97 28.9 0.2 30 4 7
sheep 1.12 −3.34 101 29.2 0.2 17 2 5
IL-12B cattle 86.5 0.8 −3.32 97 27.3 0.4 95 8 1
goat 1.70 −3.28 95 35 0.6 <1 - 2
sheep 1.59 −3.31 95 33 0.5 2 0.6 5
INF- γ cattle 80.2 1.27 −3.449 95 27.8 0.0 79 2 5
goat 1.63 −3.309 95 28.3 0.1 68 5 6
sheep 1.02 −3.409 96 27.2 0.2 128 18 2
TNF- α cattle 84.5 2.18 −3.417 91 25.0 0.3 837 192 2
goat 0.38 −3.350 99 25.1 0.1 387 35 3
sheep 0.04 −3.350 97 25.7 0.2 307 35 1
  1. Abbreviations. E, reaction efficiency; Tm, melting temperature; SD: Standard deviation; LOD, limit of detection; nc: number of copies. Abbreviations for cytokine genes see Table 1.
  2. Inter-assay variability (R) is the variation in amplification results between different runs for each sample. R was determined with amplification of cDNA synthesized from Tempus™ whole blood total RNA, in duplicate, repeated in three independent runs. R is expressed in Cq and mean number of copies (nc) with standard deviation (SD).
  3. Limit of detection (LOD) is the lowest number of copies correctly amplified, quantified that led to single specific product amplification at the expected Tm after serial dilutions of cDNA pool synthesized from the TempusTM total RNA. Nc was calculated using the following equation: Nc=(Relative amount of target (in ng) × 10−9)/(DNA length (dp) x 650) × 6.022 × 1023. 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).