Overall experimental design. a) A well established animal model of LPS- induced bladder inflammation was used. b) RNA was extracted from isolated detrusor muscle and mucosa layers. c) Extracted RNA was used to generate 6 different libraries by suppressive subtraction hybridization (SSH) in order to determine bladder tissue- and treatment- dependent transcripts. d) Transcripts were then screened and e) Sequenced f) All unique transcripts were fully annotated by querying public (PubMed, Gene Ontology, Mouse Genome ) and private (Transfac professional ) databases. g) The accession number of each SSH-selected transcript was uploaded into the PAINT 3.3 feasnet builder  to query the Transfac database . h) A regulatory network for each library was originated by a combination of SSH-selected transcripts and over-represented TF (0 < p < 0.05) in the matrix when compared to the PAINT database reference equivalent to the all the genes in the Ensembl annotated genome (Figures 2, 3, 4, 5, 6, 7). i) Unique clones were validated by QRT-PCR.
Saban et al. BMC Physiology 2006 6:1 doi:10.1186/1472-6793-6-1