Atwood et al. BMC Genomics 2011, 12:14 http://www.biomedcentral.com/1471-2164/12/14 cells expressed the highest total number of GPCRs (108) followed by N18 (105), AtT20 (79), and HEK293 cells (73). BV2 cells had the greatest total abundance of Class A receptors (88), but had equal numbers of Class A orphan GPCRs as N18 cells (figure 3). On the other hand, HEK293 cells have mRNA for the greater number of Class B and Class C orphans (figure 4). It is interest- ing that the BV2 and the N18 cell lines, which are more often used as models for specific cell types, express mRNA for the most diverse set of receptors. This pre- sumably results in a greater sensitivity to a broad spec- trum of ligands and the large number of signaling proteins for which we measured high mRNA levels in these two cell lines attest to this. It is also of interest that HEK293 and AtT20 cells have the lowest numbers of GPCRs of these four lines. Low expression (both of amount and type) may allow these cell lines to serve as a better-differentiated cell model, and, from an experi- mental perspective, may make a given cell line more attractive as a ‘clean slate’ into which gene products of interest can be introduced. mRNA expression levels do not guarantee that a spe- cific mRNA is properly translated, folded, and trafficked to produce functional protein. Discrepancies between our findings and those that have been reported using PCR-based methods may be due to differences that appear in cell lines with time, passage number and growth conditions. Discrepancies may also be attributed to the PCR-based techniques generally employed to extract and amplify mRNA: in some cases the techni- ques used in many of the studies cited here, some of which do not allow for relative quantification, may have detected very low levels of receptor mRNA that did not reach our threshold for statistical significance. In light of the potential discrepancies between microarray and PCR-based techniques, we employed qPCR analysis to test the validity of our microarray results (figure 7). The overall consistency between our qPCR results and microarray data lend confidence to the concept that the microarray analyses accurately reflect the presence of mRNA for these GPCRs and their signaling proteins. Conclusions In conclusion, this microarray analysis of the nearly full complement of non-chemosensory GPCRs and related signaling proteins in these four cell lines greatly expands our understanding of their potential signaling repertoire. These cell lines are used frequently as models of specific cell types, in heterologous expression, and as controls for antibody screening. The present work provides accessible profiles of mRNA expression that we hope will assist investigators of the GPCR superfamily in choosing the appropriate cell line to study a particular GPCR or related protein of interest and further to better Page 11 of 14 understand potential functional interactions between GPCRs and related proteins in these lines. It is our hope that this work can eventually be extended to other com- monly used cell lines such as CHO and COS7 cells when their host genomes (in this case hamster and green monkey, respectively) have been sequenced and made available for gene microarray analysis. Methods Cell cultures Human Embryonic Kidney (HEK) (catalog #CRL-1573) and AtT20 (catalog #CRL-1795) cells were purchased from American Type Culture Collection (Boston, MA). BV2 microglia cells were obtained from Dr. Nephi Stella (University of Washington) [32,33]. N18 neuroblastoma cells were obtained from Dr. William Catterall (Univer- sity of Washington) [75]. Dulbecco’s modified Eagle’s medium (DMEM), penicillin, streptomycin, and fetal bovine serum (FBS) were purchased from Gibco Life Technologies (Rockville, MD). All cell lines were grown in DMEM with 10% FBS, 100 units ml-1 penicillin, and 100 μg ml-1 streptomycin at 37°C in 5% CO2 humidified air. Importantly, BV2 cells were maintained in condi- tions that push BV2 cells towards a resting, non-acti- vated state. Cell preparation, mRNA extraction & microarray analysis Hybridization and sample imaging/quantification was performed by Indiana University’s Center for Genomics and Bioinformatics. To study mouse and human mRNA expression, we used Nimblegen 4 × 72 k arrays (NCBI MM8, and NCBI HG18, respectively; 60 mer probe lengths). These are four-plex arrays that allow for the examination of mRNA expression of ~20,000 genes from four samples. The layout provides for three probes per target, the values of which we averaged to obtain a sample value. We cultured four 10 cm plates of each cell line, performing separate extractions when the cells grew to confluence. Total RNA was extracted from cells using TRIzol reagent (Invitrogen) under RNase-free con- ditions, followed by chloroform treatment (100 ul/ml). After centrifugation, the aqueous phase containing total RNA is then extracted and treated with isopropanol (500 uL/mL) for 5-10 mins. After centrifugation, the RNA pellet was isolated, washed with ethanol (75%; 1 ml/1 mL of reagent solution), followed by another cen- trifugation and air-dried. The RNA pellet was resus- pended in RNase-free water. Total RNA quantity was assessed using NanoDrop ND-1000, and the Bioanalyzer 2100 determined the 28S/18S ratio for quality assessment. To synthesize targets for hybridization, we began with 10.0 μg of total RNA and used Invitrogen’s SuperScript Double-Stranded cDNA Synthesis kit using oligo (dT) primer (Promega) followed by DNA labeling using 1 O.D.