Annealing temperatures and qPCR efficiency were optimized with PCR products using E. coli genomic DNA as template. The

16S rRNA gene was selected as the A-1155463 price housekeeping gene. The amplification efficiency for target genes was near 100% and within 5% of the housekeeping gene of 16S rRNA. Total RNA from sorted and unsorted E. coli cells were reverse transcribed to cDNA using a reverse transcription kit (Applied Biosystems, Carlsbad, CA). cDNA was diluted 10- and 100-fold and 1 μl was assembled for qPCR reactions using the SYBR Green PCR Master Mix (Applied Biosystems, Carlsbad, selleck chemical CA). Differential expression of the same gene in sorted and unsorted E. coli was calculated with the ΔΔCt method from four replicates. The PCR program included a cycle of 95°C for 10 min, 35 cycles of 30 seconds at 94°C, 30 seconds at the optimized annealing temperature for each set of specific primers and 30 seconds at 72°C, and a melting curve analysis from 60°C to 95°C at the end. Acknowledgements This study was supported by the US National Science Foundation Biocomplexity GEN-EN Program (Grant No. BES-0412618). Plasmid pBPF-mCherry was kindly provided by Dr. Wilbert Bitter (Leiden University, the Netherlands).

Electronic supplementary material Additional file 1: Full list of genes differentially expressed in sorted E. coli cells. Full list of genes of E. coli differentially expressed in IMS sorted E. coli cells versus unsorted E. coli cells in two independent microarray studies I and II. (PDF 98 KB) Additional file 2: qPCR primers for nine tested genes.

List of primers and their optimized annealing temperatures used AP26113 in qPCR to confirm differential expression in IMS sorted versus unsorted E. coli cells. (PDF 74 KB) References 1. De Vriendt K, Theunissen S, Carpentier W, MTMR9 De Smet L, Devreese B, Van Beeumen J: Proteomics of Shewanella oneidensis MR-1 biofilm reveals differentially expressed proteins, including AggA and RibB. Proteomics 2005,5(5):1308–1316.PubMedCrossRef 2. Watnick P, Kolter R: Biofilm, city of microbes. J Bacteriol 2000,182(10):2675–2679.PubMedCrossRef 3. Whiteley M, Ott JR, Weaver EA, McLean RJ: Effects of community composition and growth rate on aquifer biofilm bacteria and their susceptibility to betadine disinfection. Environ Microbiol 2001,3(1):43–52.PubMedCrossRef 4. An D, Danhorn T, Fuqua C, Parsek MR: Quorum sensing and motility mediate interactions between Pseudomonas aeruginosa and Agrobacterium tumefaciens in biofilm cocultures. Proc Natl Acad Sci USA 2006,103(10):3828–3833.PubMedCrossRef 5. Nielsen AT, Tolker-Nielsen T, Barken KB, Molin S: Role of commensal relationships on the spatial structure of a surface-attached microbial consortium. Environ Microbiol 2000,2(1):59–68.PubMedCrossRef 6. Mashburn LM, Jett AM, Akins DR, Whiteley M: Staphylococcus aureus serves as an iron source for Pseudomonas aeruginosa during in vivo coculture. J Bacteriol 2005,187(2):554–566.PubMedCrossRef 7.