BMC Genomics 2004, 5:46 http://www.biomedcentral.com/1471-2164/5/46 concentration. An additional difference in Ct values (∆Ct) was calculated for each gene by taking the triplicate ∆Ct values for each gene in the mutant LAPN1-E266I cells and subtracting the mean ∆Ct of the triplicates for the wild- type LAPN cells. The relative expression levels were calcu- lated as = 2 -∆∆Ct [55]. Quantitative RT-PCR analysis Relative quantitative RT-PCR was preformed using Quan- tumRNA™ 18S internal standards from Ambion, Inc. (Austin TX) that included 18S Primers and Competim- ers™. By optimizing the assay and choosing an for appropriate18S Primer:Competimer ratio for each sam- ple, the 18S signal was reduced to the same linear range as that identified empirically for the gene specific product. The amplicon for the 18S primers was 315 bp. Custom primers specific to the genes of interest were synthesized by Life Technologies. The forward and reverse primers for calpactin I light chain (GenBank accession no. M16465) were 5'-GTGGACAAAATAATGAAGGAC-3' and 3'-ACAA- GAAGCAGTGGGGCAGAT-5', respectively, correspond- ing to an amplicon of 222 bp. The forward and reverse primers CEBPδ (GenBank accession no. NM_007679.1) were 5'-ATACCTCAGACCCCGACAGCG- 3' and 3'-CAAAAGTCTGTCGGAAATGTC-5', respectively, corresponding to an amplicon of 220 bp. Total RNA was isolated from LAPN1 and LAPN1-E266I cells in four sep- arate experiments, using Qiagen's RNeasy® midi kit. Reverse transcription of total RNA from each sample was carried out using random decamers and the RETROscript™ kit from Ambion. RT reactions were then subjected to PCR using the gene specific primers above (final concentration of 0.4 uM each), the appropriate 18S Primer:Competimer ratio and 10uCi/ul [α-32P]dCTP for labeling. PCR was conducted using the following cycle parameters: (94°C 30 sec, 57°C 30 sec, 72°C 30 sec) × 21 cycles for CEBPδ and 19 cycles for calpactin I light chain. Empirically derived 18S Primer:Competimer ratios were 1:18 for CEBPδ and 2:8 for calpactin I light chain. Zymography Activity of type IV collagenase (MMP9) was determined by zymography, as previously described [56]. Abbreviations Ezrin, radixin, moesin (ERM); Intracellular pH (pHi), Na- H exchanger type 1 (NHE1); NGAL (Neutrophil Gelati- nase Associated Lipocalin); type IV collagenase (MMP-9). Authors' contributions LP participated in the design of the study, prepared and analyzed samples for array analysis, conducted analyses for protein and RNA expression, and performed the statis- tical analysis. DB conceived of the study, conducted anal- yses for protein expression, and participated in the design and coordination of the study. LP and DB prepared the manuscript. 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