Atwood et al. BMC Genomics 2011, 12:14 http://www.biomedcentral.com/1471-2164/12/14 CY3-labeled random nonamer primer (TriLink Bio- technologies) and 100 U Klenow fragment 3’ > 5’ exo- (NEB) per 1.0 μg double-stranded cDNA. Single-color hybridization, post-hybridization washing and scanning were done with NimbleGen reagents and according to the NimbleGen’s User’s Guide for Expression Analysis. Images were acquired using a GenePix 4200A scanner with GenePix 6.0 software. The data from these arrays were extracted using the software NimbleScan 2.4 (Roche NimbleGen) and processed into PAIR files. We used two separate NimbleGen high-density arrays for expression analysis (design name = HG18 60 mer expr 4plex and design name = MM8 60 mer expr X4). The 4-plex arrays each consisted of 4 × 72,000 isother- mal long-oligo nucleotide probes that are 60 nucleotides in length. The first was based on HG18, Build 36 ver- sion from NCBI of Homo sapiens gene catalog, which represented 24,000 protein-encoding genes with 3 probes per target and 1 replicate, and the second was MM8 version from NCBI of Mus musculus gene catalog, which represented 18,869 protein-encoding genes with 3 probes per target and 1 replicate. Quantitative polymerase chain reaction analysis Primers for selected GPCRs and enzymes were designed using Primer-Blast (http://www.ncbi.nlm.nih.gov/tools/ primer-blast). The mouse gene was used as template in the primer design, and either mouse or human genome was used to screen for non-specific targets. Suitable pri- mers were determined in this way and were commercially produced (Integrated DNA Technologies, Coralville, Iowa). AtT20, BV-2, HEK293 and N18 cell lines were grown on 10 cm dishes until each monolayer was 50-95% confluent. Total RNA was prepared and reverse tran- scribed using a Retroscript Kit as per manufacturer’s pro- tocol (Cat# AM1710; Ambion Inc. Austin, Texas). Two micrograms of total RNA from each line was reverse transcribed, samples were diluted and qPCR was per- formed using a Stratagene Mx3000P thermocycler and Sybr Green/Low Rox polymerase mix (Cat.# 95056-100, Quanta Biosciences, Gaithersburg, MD). In the case of N18, the “no RT” control showed significant genomic DNA contamination. To compensate for this, the total RNA for this sample was treated with DNase 1 for 1 hour and again purified just prior to RT. Primers for glu- taraldehyde-3-phosphate dehydrogenase (GADPH) were used to make an internal control for each cell line with the threshold cycle set at 16. Once the standard critical threshold (Ct) was set, the relative expression levels for genes within each cell line were determined. Database information All data is MIAME compliant and has been deposited in GEO (accession number GSE25901). Page 12 of 14 Statistical analysis For statistical analysis of the microarray data, we first transformed the values from the microarray into log2 format for ease of comparison and data representation [76]. We then obtained values for 10-11 assorted semi- nal proteins that were unlikely to be expressed in these cell lines. For the human-derived HEK293 cells these were: PYY2, PSP94, SPATA3, SPATA4, SPATA5, SPATA13, SPATS1, SPESP1, SPERT, ODF1, and SPAM1. For the three murine cell lines we selected: Svs2, Svs3, Svs5, Svs6, Svs7, Sva, Svp2, Sval1, Sval2, Sval3. We averaged the expression level of each of these proteins for each of the four plates to determine the mean and standard deviation of their expression. We took these mean values to represent zero expression. To determine statistically significant expression, we added the mean plus two standard deviations of the expression levels of these seminal proteins for each plate. We then subtracted this value from the value for each receptor and protein for which we obtained a signal. The mean plus two standard deviations was considered to be the baseline for our proteins of interest as any values at this level or higher would be considered statistically signifi- cant (95% confidence that the true mean of the seminal protein expression would fall within the mean plus two standard deviations). Thus any protein remaining with a positive expression value is considered to be significantly expressed [76]. All graphs and statistical analyses were generated using GraphPad Prism 4.0 software (Hearne Scientific Software, Chicago, IL). Abbreviations CRF: corticotropin releasing factor; DMEM: Dulbecco’s modified Eagle’s medium; FBS: fetal bovine serum; GPCR: G Protein Coupled Receptor; GRK: G protein coupled receptor kinase; HEK: Human Embryonic Kidney; mGluR: metabotropic glutamate receptor; PCR: polymerase chain reaction; RGS: Regulator of G protein signaling. Acknowledgements This work was supported by the NIH (grants DA11322, DA21696, DA24122, NIH/NCRR RR025761), the Center for Genomics and Bioinformatics at Indiana University and the Indiana METACyt Initiative of Indiana University, through a major grant from the Lilly Endowment, Inc. Author details 1Department of Psychological & Brain Sciences, The Gill Center for Biomolecular Science, Indiana University, Bloomington, Indiana, USA. 2Center for Genomics and Bioinformatics, Indiana University, Bloomington, Indiana, USA. 3Graduate Program in Neurobiology and Behavior, University of Washington, Seattle, Washington, USA. Authors’ contributions BKA carried out the drafting of the manuscript and the statistical analysis. JL performed the microarray analysis and assisted in drafting the manuscript. JWM performed the cell line preparation, mRNA extraction and qPCR experiments. KM participated in the design and coordination of the study. AS conceived of the study, participated in the design and coordination of the study, critical review of the manuscript, and performed the data extraction. All authors contributed to writing, reading, and approving the final manuscript.