. Author manuscript; available in PMC: 2014 Dec 15.

Published in final edited form as: Vet Immunol Immunopathol. 2013 Dec 15;156(0):229–234. doi: 10.1016/j.vetimm.2013.09.019

Table 2.

Assay amplification efficiency, slopes of regression lines, and inter-assay and intra-assay variation for reference gene and cytokine genes

Gene	Efficiency (%)	Amplification Factor	Slope	Inter-assay Variation		Intra-assay Variation
Gene	Efficiency (%)	Amplification Factor	Slope	CV (%)	SD (C_t)	CV (%)	SD (C_t)	SD Range
GAPDH	92.86	1.93	-3.51	7.07	1.22	0.24	0.04	0.015 - 0.091
IL-2	90.33	1.90	-3.58	6.97	2.13	0.33	0.098	0.037 - 0.244
IFN-γ	90.27	1.90	-3.58	4.73	1.49	0.88	0.277	0.039 - 0.382

Assay efficiency for reference gene and cytokine genes determined using five tenfold dilutions (10¹-10⁶) of total RNA isolated from a healthy intact female Walker hound

Inter-assay variation determined by running one sample in triplicate on nine different days. CV was calculated using the mean and standard deviation of C_t values for one sample run on nine different days.

Intra-assay variation was calculated using the mean and standard deviation of C_t values for a reaction run in triplicate. This was replicated on nine different plates all using the same RNA sample, and the CVs calculated for each run were averaged together.

CV, coefficient of variation SD, standard deviation