deposited data can be found in the gene expression omnibus ( geo ) database : http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gse57560 .
gse57560  correlation of gene expression with bladder capacity in interstitial cystitis / bladder pain syndrome .
a flow diagram outlining of the experimental design for this study can be found in fig .  1 .
experimental biopsy tissue was collected either during cystoscopy or at surgery ( for patients who were undergoing cystectomy for end stage disease ) under general anesthesia from patients diagnosed with interstitial cystitis / bladder pain syndrome ( ic / bps ) , , , , .
cystoscopy patients first underwent hydrodistention at 100  ml of h2o for a period of 5  min .
study biopsies were taken post hydrodistention from the posterior bladder wall using a cold - cup technique and a portion of each biopsy specimen was sent for normal clinical analysis by the wake forest medical center pathology department ( following standard hospital protocol ) .
the remaining sample was immediately submerged in 200  l of rnalater and stored at   20  c until processing . for patients undergoing cystectomy ,
an amount of tissue similar to that which would be collected at biopsy was harvested from the posterior bladder through a single scalpel incision and submerged in 200  l of rnalater and stored at   20  c in a similar fashion .
bladder capacity data and cystoscopic findings for these subjects were retrieved from the patient 's last cystoscopy recording in the medical record and included in this analysis ( table  1 ) . for the purposes of this study ,
low bladder capacity was defined as <  400  ml ( volume at hydrodistention ) .
control tissue was likewise collected during cystoscopy . as these patients did not have any clinical indication for the performance of hydrodistention ,
biopsy tissue was homogenized by sonication and total rna was extracted using rneasy minelute plus columns ( includes on - column dnase digestion ) and reagents ( qiagen , valencia , ca ) according to the manufacturer 's instructions .
rna was eluted from the columns in nuclease - free water and rna quantity and purity was determined spectrophotometrically on a nanodrop nd-1000 ( nanodrop technologies , wilmington , de ) by measuring absorbance at 260/280  nm ( mean for all samples  =  2.09    0.02 ) and rna quality was determined using an agilent bioanalyzer ( agilent technologies , inc . , palo alto , ca ; mean rin  =  9.4    0.24 for all samples ) .
total rna was shipped on dry ice to a microarray core facility ( city of hope functional genomics core , duarte , ca ) for processing .
cyanine-3 ( cy3 ) labeled crna was prepared from 0.5  g total rna using the one - color low rna input linear amplification plus kit ( agilent ) according to the manufacturer 's instructions , followed by rnaeasy column purification ( qiagen , valencia , ca ) .
1.5  g of cy3-labeled crna ( specific activity >  10.0  pmol  cy3/g crna ) was fragmented at 60  c for 30  min in a reaction volume of 250  ml containing 1 agilent fragmentation buffer and 2   agilent blocking agent following the manufacturer  's instructions . on completion of the fragmentation reaction , 250  ml of 2 agilent hybridization buffer was added to the fragmentation mixture and hybridized to sureprint human gene expression v2 microarrays ( agilent technologies ) containing 60-mers for 50,599 biological features for 17  h at 65  c in a rotating agilent hybridization oven .
after hybridization , microarrays were washed 1  min at room temperature with ge wash buffer 1 ( agilent ) and 1  min with 37  c ge wash buffer 2 ( agilent ) , then dried immediately by brief centrifugation
. slides were scanned immediately after washing on the agilent dna microarray scanner ( g2505b ) using one color scan setting for 1    44  k array slides ( scan area 61    21.6  mm , scan resolution 10  m , dye channel is set to green and green pmt is set to 100% ) .
the scanned images were analyzed with feature extraction software 9.1 ( agilent ) using default parameters ( protocol ge1-v1_91 and grid : 012391_d_20060331 ) to obtain background subtracted and spatially de - trended processed signal intensities .
the anonymized clinical data were deposited in the gene expression omnibus database ( geo http://www.ncbi.nlm.nih.gov/geo/ under accession number gse57560 ) . following log transformation of the processed signal intensity data , principle component analysis ( pca ) and unsupervised hierarchical clustering
were performed ( qlucore omics explorer ) to determine the similarity among samples within and between groups ( fig .  2 ) .
further statistical analyses to measure differentially expressed transcripts ( dets ) unique to each phenotype were then performed by applying student 's t - test ( @fold change    1.5 ; p    0.05 ) for the pair - wise comparison ( genesifter analysis edition 4.0 ) using benjamini and hochberg fdr correction .
the list of dets generated from the pair - wise comparison ( e.g. low bladder capacity versus normal bladder capacity ) with an adjusted p    0.05 was imported into ingenuity pathway analysis software for gene ontology and pathway analysis .
a flow diagram outlining of the experimental design for this study can be found in fig .  1 .
experimental biopsy tissue was collected either during cystoscopy or at surgery ( for patients who were undergoing cystectomy for end stage disease ) under general anesthesia from patients diagnosed with interstitial cystitis / bladder pain syndrome ( ic / bps ) , , , , .
cystoscopy patients first underwent hydrodistention at 100  ml of h2o for a period of 5  min .
study biopsies were taken post hydrodistention from the posterior bladder wall using a cold - cup technique and a portion of each biopsy specimen was sent for normal clinical analysis by the wake forest medical center pathology department ( following standard hospital protocol ) .
the remaining sample was immediately submerged in 200  l of rnalater and stored at   20  c until processing . for patients undergoing cystectomy ,
an amount of tissue similar to that which would be collected at biopsy was harvested from the posterior bladder through a single scalpel incision and submerged in 200  l of rnalater and stored at   20  c in a similar fashion .
bladder capacity data and cystoscopic findings for these subjects were retrieved from the patient 's last cystoscopy recording in the medical record and included in this analysis ( table  1 ) . for the purposes of this study ,
low bladder capacity was defined as <  400  ml ( volume at hydrodistention ) .
control tissue was likewise collected during cystoscopy . as these patients did not have any clinical indication for the performance of hydrodistention ,
biopsy tissue was homogenized by sonication and total rna was extracted using rneasy minelute plus columns ( includes on - column dnase digestion ) and reagents ( qiagen , valencia , ca ) according to the manufacturer 's instructions .
rna was eluted from the columns in nuclease - free water and rna quantity and purity was determined spectrophotometrically on a nanodrop nd-1000 ( nanodrop technologies , wilmington , de ) by measuring absorbance at 260/280  nm ( mean for all samples  =  2.09    0.02 ) and rna quality was determined using an agilent bioanalyzer ( agilent technologies , inc . , palo alto , ca ; mean rin  =  9.4    0.24 for all samples ) .
total rna was shipped on dry ice to a microarray core facility ( city of hope functional genomics core , duarte , ca ) for processing .
cyanine-3 ( cy3 ) labeled crna was prepared from 0.5  g total rna using the one - color low rna input linear amplification plus kit ( agilent ) according to the manufacturer 's instructions , followed by rnaeasy column purification ( qiagen , valencia , ca ) .
1.5  g of cy3-labeled crna ( specific activity >  10.0  pmol  cy3/g crna ) was fragmented at 60  c for 30  min in a reaction volume of 250  ml containing 1 agilent fragmentation buffer and 2   agilent blocking agent following the manufacturer  's instructions . on completion of the fragmentation reaction , 250  ml of 2 agilent hybridization buffer was added to the fragmentation mixture and hybridized to sureprint human gene expression v2 microarrays ( agilent technologies ) containing 60-mers for 50,599 biological features for 17  h at 65  c in a rotating agilent hybridization oven .
after hybridization , microarrays were washed 1  min at room temperature with ge wash buffer 1 ( agilent ) and 1  min with 37  c ge wash buffer 2 ( agilent ) , then dried immediately by brief centrifugation
. slides were scanned immediately after washing on the agilent dna microarray scanner ( g2505b ) using one color scan setting for 1    44  k array slides ( scan area 61    21.6  mm , scan resolution 10  m , dye channel is set to green and green pmt is set to 100% ) .
the scanned images were analyzed with feature extraction software 9.1 ( agilent ) using default parameters ( protocol ge1-v1_91 and grid : 012391_d_20060331 ) to obtain background subtracted and spatially de - trended processed signal intensities .
the anonymized clinical data were deposited in the gene expression omnibus database ( geo http://www.ncbi.nlm.nih.gov/geo/ under accession number gse57560 ) .
following log transformation of the processed signal intensity data , principle component analysis ( pca ) and unsupervised hierarchical clustering were performed ( qlucore omics explorer ) to determine the similarity among samples within and between groups ( fig .  2 ) .
further statistical analyses to measure differentially expressed transcripts ( dets ) unique to each phenotype were then performed by applying student 's t - test ( @fold change    1.5 ; p    0.05 ) for the pair - wise comparison ( genesifter analysis edition 4.0 ) using benjamini and hochberg fdr correction .
the list of dets generated from the pair - wise comparison ( e.g. low bladder capacity versus normal bladder capacity ) with an adjusted p    0.05 was imported into ingenuity pathway analysis software for gene ontology and pathway analysis .
we demonstrate that low capacity and normal capacity ic / bps bladders have significantly different molecular characteristics , and this difference may reflect a fundamental difference in disease processes .
meanwhile , ic / bps patients with normal bladder capacity exhibit similar molecular profiles to control subjects .
given the promising results of this pilot study we are conducting further research into the correlation between molecular and clinical findings and the development of an ic / bps biomarker .
 interstitial cystitis and bladder pain syndrome ( ic / bps ) are terms used to describe a heterogeneous chronic pelvic and bladder pain disorder . despite its significant prevalence , 
 the disease etiology is not well understood and providing diagnosis and treatment can be challenging . in our study , 
 published recently in the journal of urology ( colaco et al . , 2014 ) , we describe the use of microarrays as a tool to characterize ic / bps and to determine if there are clinical factors that correlate with gene expression . 
 this data - in - brief article describes the methodology for that study , including data analysis , in further detail . 
 deposited data can be found in the gene expression omnibus ( geo ) database : gse57560 . 
Direct link to deposited data
Experimental design, materials and methods
Sample procurement
RNA extraction and QC
Gene expression analysis by microarray
Data processing for gene expression and gene ontology/pathway analysis
Discussion
Conflict of interest