Results
We found that the EpiQuick Total Histone Extraction Kit (EpiGentek) and the Total Histone H3 Acetylation Detection Fast Kit (EpiGentek) were effective in house sparrows. Estimates of H3 acetylation ranged from 0.01 to 2.08 OD/mg/ml in liver and from 0.08 to 2.18 OD/mg/ml in spleen.CV among replicates ranged from 5.0 to 48.2% in liver and 3.0 to 46.6% in spleen. Our criteria of CV 25%, or higher, identified 3 outlier estimates in liver and 7 outlier estimates in spleen. TheCV among replicates was much lower than the CV among individuals before and after removing outliers. Among replicates before removing outliers, CV was 16.9% for liver and 26.5% for spleen, and CV among individuals within tissue was 49.9% for liver and 65.5% for spleen. Among replicates after removing outliers, CVwas 12.2% for liver and 10.7% for spleen, and among individuals was 51.1% for liver and 66.1% for spleen. There were no correlations between CV (among replicates and after removing outliers) and tissue mass (Liver: r = -0.09, P = 0.38, Spleen: r= 0.17, P = 0.29), histone concentration (Liver: r = 0.30,P = 0.16, Spleen: r = 0.26, P = 0.20), or mean H3 (Liver: r = -0.45, P = 0.06, Spleen: r = -0.03,P = 0.46).
We detected high level of variation among individuals (Table 1). Mean H3 histone acetylation in liver was 3.82 OD/mg/ml (σ = 2.58); in spleen, mean H3 histone acetylation was 3.51 OD/mg/ml (σ = 2.45). We failed to detect significant differences between tissues (t-test P = 0.38,f-test P = 0.87). We caution that based on the high variation observed, these results should not be interpreted as indicating no differences exist in H3 acetylation among tissue, rather that they indicate high variation among individuals.
Interestingly, house sparrows with higher epigenetic potential had higher H3 acetylation in liver tissue (High EP Mean H3AC = 4.13 OD/mg/ml; Low EP Mean H3AC = 1.56 OD/mg/ml;t-test = 0.046), and a similar trend occurred in spleen (High EPMean H3AC = 4.04 OD/mg/ml; Low EP Mean H3AC = 1.78 OD/mg/ml; t-test = 0.108; Figure 1). Also, we found a negative correlation between H3 acetylation in spleen and TLR-4 expression in cecum among house sparrows (r = -0.595, P = 0.02).