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Meanwhile, nine miRNAs responded exclusively to NE stimulation including five (cgi-miR-190, cgi-miR-133, ci-miR-278, C24628_29044 and scaffold659_26519) up-regulated and four (cgi-miR-182, cgi-miR-92d, scaffold1600_212, and scaffold508_4253) down-regulated.
Among them, two ACh-responsive miRNAs (cgi-miR-125 and cgi-miR-8) and six NE-responsive miRNAs (cgi-miR-125, cgi-miR-133, cgi-miR-190, cgi-miR-278, cgi-miR-8 and cgi-miR-92d) were also found as immune-responsive [ 18].
On the other hand, 21 miRNAs exhibited different expression pattern after ACh or NE stimulation, with six of them (cgi-miR-182, cgi-miR-242, scaffold1600_212, cgi-miR-1989, scaffold1785_16815 and scaffold625_3183) in an opposite trend.
Five miRNAs (cgi-miR-125, cgi-miR-8, scaffold1144_2255, scaffold1711_599 and scaffold226_18954) were found responsive to both the ACh and NE stimulation.
Prior to building the classifier we calculated expression of the selected genes relative to the median expression the reference genes as measured on the same microarray (rps19, cgi- 119, ctbp1 and b2m were used as the reference genes) [ 8, 9].
For example, five out of 38 neurotransmitter-responsive miRNAs (cgi-miR-125, cgi-miR-8, scaffold1144_2255, scaffold1711_599 and scaffold226_18954) showed similar expression pattern after ACh or NE stimulation, and immune-related GO terms including response to stimulus and gene expression were annotated from their target genes (Fig. 10a).
In the RFC1 gene, CpG sites 15 17 in the 5′ shore region and sites 18 30 in the CpG island 1 (CGI-1) showed a statistically significant (p < 0.05) lower methylated fraction in blood leukocytes.
In our analysis we used only protein coding genes and divided them into two classes: (1) CGI-genes, where the 5'end is found within a CpG island; (2) And nonCGI-genes, where 5'end is not located in a CpG island (see Additional file 11).
