BMC Genomics 2004, 5:46 http://www.biomedcentral.com/1471-2164/5/46 Methods Cell culture and RNA preparation The generation of LAPN and LAPE cells was as previously described [5]. In brief, NHE1-null LAP1 cells developed from parental Ltk-mouse muscle fibroblasts [16] were used for stable expression of wild-type NHE1 (LAPN cells) or expression of NHE1-E266I containing a single point substitution of glutamate266 for isoleucine that results in complete loss of ion translocation activity (LAPE) [5,6]. Cells were maintained in DMEM supplemented with 10% FCS in the presence of 25 mM NaHCO3 and 5% CO2. Total RNA was prepared from cells plated for 48 h by using Qiagen's RNeasy® midi kit.RNA. RNA was collected from five independent cell platings and used for five sep- arate DNA array hybridizations. cDNA synthesis and microarray hybridization Total RNA was converted to double-stranded cDNA using the SuperScript Choice system (Gibco BRL), except that HPLC-purified T7-(dT)24 oliomer (5'-GGCCAGTGAATT- GTAATACGACTCACTATAGGGAGGCGG – (dT)24) was used instead of the oligo (dT) or random primers pro- vided with the SuperScript Choice kit. Double-stranded cDNA was collected by ethanol precipitation. Biotinylated cRNA was then generated from the cDNA by an in vitro transcription (IVT) reaction using the ENZO BioArray™ HighYield™ RNA Transcript Labeling Kit. IVT products (cRNA) were collected by using Qiagen's RNeasyR mini kit, then ethanol-precipitated and quantitated. The cRNA was fragmented by alkaline treatment and hybridized to a GeneChip probe array from Affymetrix (Santa Clara, CA). The Affymetrix murine oligonucleotide array (Mu11KSubB) is complementary to ~6,500 murine genes and expressed sequence tags (ESTs). Each gene or EST is represented on the array by 16 – 20 feature pairs. Each fea- ture pair contains a 25-bp oligonucleotide sequence, which is either a perfect match to the gene or a single cen- tral-base hommomeric mismatch control. Microarray hybridization analysis Affymetrix GeneChip analysis was performed using stand- ard procedures [54]. The expression level of any particular transcript was calculated by subtracting he difference between the fluorescence intensities of the perfect match and mismatch feature pairs and then averaging over the entire probe set (Avg Diff). The Avg Diff value for each transcript was averaged over the five experiments for LAPN and LAPE cells; these average values obtained from five independent hybridizations were then used to calcu- late fold changes in LAPE cells relative to LAPN cells for each transcript. We did not use comparison algorithms supplied with the Affymetrix software. Immunoblot analysis Proteins from total cell lysates were separated by SDS- PAGE as previously described [5] and transferred to PVD nitrocellulose membranes for immunoblotting. Antibod- ies for immunoblotting included Fyn (NeoMarkers), PLCγ-1 and Erk 1–2 (Cell Signaling), GADD45, FEN1, and cyclin B1 (Santa Cruz Biotechnology), and actin (Sigma). TaqMan™ analysis Confirmation of GeneChip data was accomplished using TaqMan™ chemistry with the ABI 7700 Prism real-time PCR instrument (ABI, Foster City CA). Custom primers specific to the genes of interest were synthesized by Life Technologies and TaqMan™ probes for each gene were synthesized by Integrated DNA Technologies, Inc. The for- ward and reverse primers for mouse GADD153 (GenBank accession no. X67083) were 5'-GAAACGAAGAGGAA- GAATCAAAAAC-3' and 5'-ATCTGGAGAGCGAG- GGCTTT-3', respectively, and the probe was 5'-FAM/ ACCCTGCGTCCCTAGCTTGGCTGAC/TAM-3', corre- sponding to an amplicon of 122 bp. The forward and reverse primers for mouse Wee1 kinase (GenBank accession no. NM_009516) were 5'-TTGCTCTTGCTCT- CACAGTCGT-3' and 5'-TGGGAAAGCACTTGTGGGAT-3', respectively, and the probe was 5'-FAM/CCTTCCCA- GAAATGGAGAGCACTGGC/TAM-3', corresponding to an amplicon of 118 bp. The forward and reverse primers for mouse Glut4 (GenBank accession no. NM_009204) were 5'-TGGCCATCTTCTCTGTGGGT-3' and 5'-ATTGGCTAG- GCCCATGAGG-3', respectively, and the probe was 5'- FAM/TATGCTGGCCAACAATGTCTTGGCC/TAM-3', cor- responding to an amplicon of 138 bp. The forward and reverse primers for mouse 24p3 (GenBank accession no. W13166) were 5'-GGCAGCTTTACGATGTACAGCA-3' and 5'-TCTGATCCAGTAGCGACAGCC-3', respectively, and the probe was 5'-FAM/CATCCTGGTCAGGGACCAG- GACCAG/TAM-3', corresponding to an amplicon of 111 bp. For each gene, PCR was conducted in triplicate with 50 µl reaction volumes of 1x PCR buffer A (Applied Bio- systems, Foster City, CA), 2.5 mM MgCl2, 0.4 µM each primer, 200 µM each dNTP, 100 nM probe and 0.025 u/ µl Taq Gold (ABI, Foster City CA). For each experiment, a large master mix of the above components was made and aliquoted into each optical reaction tube. Each primer/ probe set (5 – 10 µl) was then added, and PCR conducted using the following cycle parameters: 95°C 12 min × 1 cycle, (95°C 20 sec, 60°C 1 min) × 40 cycles. Data analy- sis was carried out using sequence detection software that calculates the threshold cycle (Ct) for each reaction which is used to quantitate the amount of starting template in the reaction. A difference in Ct values (∆Ct) was calculated for each gene by taking triplicate Ct values from three reac- tions and subtracting the mean Ct of the triplicates for the control gene, GAPDH, for each cDNA sample at the same Page 11 of 13 (page number not for citation purposes)