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

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

Target gene	Species	Tm (°C)	Intercept	Slope	E (%)	Inter-assay variability				LOD nc
Target gene	Species	Tm (°C)	Intercept	Slope	E (%)	Mean Cq	SD Cq	Mean nc	SD nc	LOD 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

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.
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).
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 Tempus^TM 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).

ISSN: 1746-6148