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).