Atwood et al. BMC Genomics 2011, 12:14 http://www.biomedcentral.com/1471-2164/12/14 Page 10 of 14 Figure 7 Quantitative polymerase chain reaction validation of microarray analysis. Quantitative PCR analysis of 10 genes from (A) HEK293, (B) AtT20, (C) BV2, and (D) N18 cell lines. mRNA levels determined from qPCR analysis were plotted against the mRNA levels measured in the microarray analyses in figures 1, 3 and 6. The dashed lines indicate the threshold of statistical significance for the microarray data. Points that fall between 0 and this threshold indicate genes that were detected with the microarray analysis but failed to meet statistical significance. Lower critical threshold (Ct) values indicate high mRNA copy numbers. regulation of G protein mediated signaling as significant levels of mRNA for beta-arrestin1 and 2, GRK3-5 and a number of RGS subtypes were seen. Much less is known of the expression patterns of GPCRs in AtT20 cells. We confirmed the presence of Sstr1 and Sstr2 mRNA in AtT20 cells [30], but did not observe significant levels of any of the other receptors that have previously been reported such as Chrm4 [51], Vipr1a, Adcyap1r1, or Sstr5 [30,52,53]. The lack of sig- nificant levels of CRF receptor mRNA was of interest as AtT20 cells have been reported to be sensitive to CRF [54]. mRNA for Hrh3 or Hrh4 histamine receptors were not detectable, despite reports of their presence [55,56], but a low level of Hrh2 was found. Tacr1 [31] was not detected, nor were Tacr2 and Tacr3. We also did not detect the orphan Cmkor1/Cxcr7 [57]. It is worth not- ing that there are at least two AtT20 clones available and this may explain some of the differences we found. Generally speaking, AtT20 cells possess a much less diverse complement of GPCR-related signaling and reg- ulatory gene products than the other cell lines tested. Of the approximately 120 GPCR signaling gene products that we studied here, AtT20 cells had 45 significantly expressed signaling related proteins compared to 72 or more in each of the 3 other cell lines. AtT20 cells were the only ones to not have significant levels of beta- arrestin1 (Arrb1) or GRK3-5. Only three RGS gene pro- ducts were found at significantly high levels: Rgs14, Rgs20, and Snx13. BV2 cells are commonly used to study microglial biol- ogy. These cells exist in a continuum between two states: a resting state and an activated state. For exam- ple, upon interferon gamma treatment (primed state), BV2 cells increase both Cnr2 and Gpr55 mRNA levels, whereas with lipopolysaccharide treatment (activated state) these levels decrease [58]. In our case, BV2 cells were cultured under conditions expected to promote a resting state. We can confirm that as reported in the lit- erature BV2 cells express Adora3 [59], Cnr2 [33,58], Mtnr1a [60], Galr2 [61], Ccr5, Cxcr3 [62] and Rgs2, 10, 12 [27,28,63]. We also detected mRNA for receptors that have been reported to be upregulated in response to stimulation such as Ccrl2 [28,64], Ptafr, Ptigr [64] and Rgs7 [65]. Here, too, we identified a few receptors that have been reported present for which we found levels of mRNA below the levels that we defined as sta- tistically significant: Hcrtr1 [66], Gpr55 [58] and the receptor for Cx3cr1 [67]. We did not detect Prokr1 which is upregulated with activation [27], nor for Oprm1, P2ry1 and Gpr149 which are downregulated fol- lowing activation [27,28]. Several reports have identified the presence of mGluRs in BV2 and primary microglia [21,68,69]. Interestingly, Grm1 is generally thought not to be present in BV2 cells, yet it was the only one that we significantly detected. We did find low, non-signifi- cant lev els of Grm3, 6, and 8. The differences may be due to differences in the activation state of the cell lines. BV2 cells appear have a diverse receptor and signaling gene product profile. Of the cell lines tested, the least well characterized is the N18 neuroblastoma line. They are known to express Cnr1 [70], which we confirmed with our analysis. It has been reported by some that they do not express mus- carinic receptors [71], but our analysis shows that they have mRNA for three different types (Chrm2, 3, and 4) consistent with other reports [72,73]. These studies also found evidence for secretin, prostacyclin, opioid and alpha2-adrenergic receptors In support of this we found significantly high levels of mRNA for Ptgir1, Ptger1, Ptger3, Ptger4, Oprd1, Oprl1 and Adra2b. There were also levels of mRNA for Sctr, Adra2a, Oprk1 and Oprm1 that did not reach statistical significance. Another report describes P2ry2 expression [74], but for this we only found low non-significant levels. Beyond these reports there seems to be little known of GPCR signaling in this cell line and this analysis substantially extends our knowledge of its complement of GPCR expression, at least at the mRNA level. Like BV2 cells, N18 cells appear to have a large repertoire of GPCRs, effectors, and regulating proteins, being consistently one of the highest expressers in each receptor class analyzed. Interestingly, even the most prolific cell line expressed only a small portion of the full repertoire of non-chemo- sensory GPCRs (~360). Of these four cell types, BV2