{"database":"biostudies-arrayexpress","file_versions":[],"scores":null,"additional":{"omics_type":["Metabolomics","Unknown","Transcriptomics","Genomics","Proteomics"],"submitter":["Mohan Pammi Venkatesh"],"study_type":["transcription profiling by array"],"organism":["Candida albicans"],"species":["Candida albicans"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/E-GEOD-35438"],"description":["To explain enhanced biofilm formation and increased dissemination of S. epidermidis in mixed-species biofilms, microarrays were used to explore differential gene expression of S. epidermidis in mixed-species biofilms. One sample from single species biofilm (S1) and mixed-species biofilm (SC2) were excluded from analyses for outliers. We observed upregulation (2.7%) and down regulation (6%) of S. epidermidis genes in mixed-species biofilms. Autolysis repressors lrgA and lrgB were down regulated 36-fold and 27-fold respectively and was associated with increased eDNA possibly due to enhanced autolysis in mixed-species biofilms. These data suggest that bacterial autolysis and release of eDNA in the biofilm matrix may be responsible for enhancement and dissemination of mixed-species biofilms of S. epidermidis and C. albicans. Staphylococcal gene expression in mixed-species biofilms with Candida and in single species biofilms of S. epidermidis were analyzed. The experiment was repeated thrice on 3 different days (3 biological replicates each for single species biofilms of S. epidermidis and mixed-species biofilms). Only 2 biological replicates were analyzed and one biological replicate was not analyzed (S1 and SC1 - raw data files are provided on the Series record). Single species biofilms of S. epidermidis (strain 1457) and C. albicans (strain 32354) and mixed-species biofilms were formed on 6-well tissue culture plates. Five ml of organism suspensions (O.D. 0.3, S. epidermidis 107 CFU/ml or C. albicans 105 CFU/ml)  or 2.5 ml each for mixed-species biofilms for 24 hr. RNA was harvested from single species and mixed-species biofilms."],"repository":["biostudies-arrayexpress"],"sample_protocol":["Growth Protocol - Single species biofilms of S. epidermidis (strain 1457) and C. albicans (strain 32354) and mixed-species biofilms were formed on 6-well tissue culture plates. Five ml of organism suspensions (O.D. 0.3, S. epidermidis 107 CFU/ml or C. albicans 105 CFU/ml)  or 2.5 ml each for mixed-species biofilms for 24 hr.","Labeling - Alexa Fluor 555 (Invitrogen A32756) was coupled to cRNA following the manufacturer’s instructions. Unincorporated dye was removed using RNeasy Mini Columns (Qiagen 74104). The Qiagen’s quick clean-up protocol was followed. cRNA Fragmentation. cRNA coupled to fluorescent dye was fragmented using proprietary technology. The expected mean fragment length is 100 -125 nucleotides. Fragmented cRNA is considered sufficiently fragmented for use on MYcroarrays if the mean fragment length less than 200 nucleotides.","Hybridization - CUST-40K-Staphylococcus_epidermidis_RP62A. Custom 40K Staphylococcus epidermidis RP62A MYcroarrays were manufactured. Each MYcroarray slide has one array composed of 40,960 spots, of which 33,715  spots contain 45mer  probes for S. epi genes, 525 features are “empty” (i.e., they contain no probe), 608 features contain MYcroarray in-house QC probes and 112 features contain positive control probes for assessing hybridization and washing stringency. There are 5 identical replicates of each S. epi probe such that a total of 6,743 S. epi genes are surveyed by each array. In addition, there are 6000 probes for randomly selected Candida genes.     Single color Hybridizations. cRNA derived from all samples was coupled to Alexa Fluor 555. Eight micrograms of each target was hybridized separately to one array.  Since the customer sent 3 biological replicates of two sample types (pure culture and cultured with Candida) each of the 6 targets was hybridized separately to one array. Hybridization was performed for >18hrs at 45oC in a proprietary hybridization buffer.  Wash and Scan Arrays. The array sandwich was opened submerged in 6X SSPE at 22oC and stored briefly (<2min) in fresh 6X SSPE until all arrays were ready to wash. The arrays were washed twice in 1X SSPE at 22oC for 3 minutes per wash while the wash solution was gently circulated over the arrays. The arrays were washed in 1X SSPE at 45C for 5 min. Finally, the arrays were allowed to cool to 22oC for 3 minutes in 1X SSPE then exposed to 0.25X SSPE for 30sec and spun dry in a microarray centrifuge. The arrays were scanned in an Axon 4000B Scanner (Molecular Devices) set at 5m per pixel resolution. The PMT setting was adjusted to appreciate the maximum dynamic range of signal. This was accomplished by increasing the PMT until a few features showed at least some saturation.","Nucleic Acid Extraction - RNA was harvested from single species and mixed-species biofilms using RNeasy Mini kit (Qiagen), a Fast-RNA Pro-BLUE kit (MP Biomedicals) according to manufacturer’s instructions (47, 49).  Microbial cells were lysed in a high-speed homogenizer (FastPrep24 instrument), and the isolated RNA was treated with RNAse-free DNAse-I. An aliquot of RNA was centrifuged and the pellet was washed with 80% ethanol. Following centrifugation, the supernatant was carefully aspirated, the residual ethanol was removed by brief drying (37oC) and the pellet was suspended in RNase free water. Total RNA was quantified with the use of a NanoDrop-1000 spectrophotometer. In addition, the integrity of the total RNA was analyzed (Agilent 2100 Bioanalyzer). Enrichment of mRNA. Ten micrograms of total RNA was converted to enriched mRNA using Applied Biosystem’s (Ambion) MicrobExpressTm Kit (AM1905). The manufacturer’s protocol was followed. Enriched mRNA was analyzed by the Agilent 2100 Bioanalyzer. The expected yield of enriched mRNA from ten micrograms of total RNA is typically 1.0 - 2.5μg.     Production of cRNA. Enriched mRNA (200ng) was converted to cRNA using Applied Biosystem’s (Ambion) MessageAmp™ II-Bacteria RNA Amplification Kit (AM1790). Amino-allyl-UTP was incorporated into the cRNA during the IVT reaction. The manufacturer’s protocol was followed. The expected yield from 200ng is about 100μg."],"figure_sub":["MIAME Score","Raw Data","Organization","Assays and Data","Processed Data","MAGE-TAB Files","Array Designs"],"pubmed_authors":["Mohan Pammi","Mohan Pammi Venkatesh"],"data_protocol":["Assay Data Transformation - ID_REF = <br>VALUE = median normalized signal intensity","Image Adquisition - Scanned images were quantified using GenePix Pro Software (version 6.1.0.4). For signal extraction, circular feature indicators (35um diameter) were centered over each spot. Median feature pixel intensity was extracted.   Saturated Spots or Spots Manually Flagged “Bad”. Some spots had some saturation due to adjusting the PMT until the full dynamic range was appreciated. Nonetheless, the PMT for this study was 375 (adjustable PMT range is 85 - 850) suggesting very robust signal while background values were no higher than 0.14% of the full dynamic range. This study, therefore, had excellent signal to background noise. Table 1 lists the frequency and percent of spots with at least 10% saturation (>=SAT10). There were so few spots with saturation that spots with saturation were permitted to be analyzed.  All spots were reviewed manually and some spots were flagged “bad” (a value of -100 in the “flags” field in the raw data [.gpr] files) due to issues such as dust, bubbles or proximity to gaskets. Table 1 reports the distribution of good (not flagged bad) spots in all probe sets (PS, each has 5 technical probe replicates; n = 6743/array). The data show that a trimmed mean (intra-array normalized signal value) was calculated for 99.77% of the probe sets (n = 26,972) in the study. A mean was used to calculate the signal value for the remaining few probes sets (n=60) because they had only 2 or 3 good probe replicates.","Feature Extraction - Due to the manufacturing process, the area immediately surrounding a spot should not be used to estimate background levels. We know that when probes do not bind target, the residual signal is very different compared with the surrounding area. A global background correction algorithm within each array is used instead of a local background correction factor. Empty” spots  was not used as a measure of background because they lack probe sequences and behave differently (to nonspecific hybridization) compared to spots containing probes.  Consequently, the background value was determined by the 5th percentile darkest feature from the distribution of median signal intensities for all Staphylococcus epidermidis RP62A spots for each array. To use local background values would over-estimate the contribution of background signal in the extracted signal intensity of each probe.     Normalization. To adjust for differences in dye incorporation, a scale factor was created to equalize signal across all arrays. Only probes that met the following criteria were used to calculate the scale factor: only S. epi spots were used; spots must have less than 10% saturated pixels; spots could not be flagged “bad”; median signal had to be more than 4 fold background; and all arrays had to have each spot qualify as a normalization spot. There were 15,554 qualifying spots. The average signal for these qualifying probes, called normalization features (NF), was calculated for each array.            Present Call. An estimation of whether or not a transcript was detected by a probe was calculated. The estimation is a “present” call. The present call is based on the trimmed mean signal for each gene relative to the global background level on each array. For each gene (probe), if the trimmed mean signal was greater than 3 times the background signal (see “Background Correction” above), then the transcript was considered “present”.  Analysis Strategy. Samples S1 and SC1 were sent separately from samples S2, S3, SC2 and SC3. Samples S1 and SC1 replaced the original samples that had failed QC. The fresh S1 and SC1 samples passed QC and were included in the study. Unfortunately, the “on-array” behavior of SC1 suggested that it was substantially different from the other samples. Table 2 lists several on-array characteristics for each sample. While S1 had a bit higher background and about 10% less genes considered to be “present”, this sample was omitted given that its matched pair SC1 had significant issues (very low signal resulting in a huge scale factor and consequently very few genes considered present); this despite the fact that both S1 and SC1 samples had very good QC parameters for dye incorporation and mean fragment size. So both SC1 and S1 were omitted from the following analysis.","Feature Extraction - Signal intensity values were extracted from scanned images using GenePix® Pro 6 software (Molecular Devices). Briefly, a 30-micron circular feature indicator was centered on each spot and median feature pixel intensity was extracted. Saturated signals were replaced by lower PMT scan values. The raw gpr files were loaded in Genespring GX 11.5, the data log2 transformed; background corrected, and normalized using the Quantile algorithm. Hierarchical clustering map was generating using Euclidean algorithm with the average linkage rule. Differential gene expression between the two samples groups (S. epidermidis and mixed-species biofilms) was evaluated by unsupervised unpaired t-test on the log2 transformed mean data. A fold-change ratio (mixed-species biofilms vs. S. epidermidis biofilms) was calculated with a fold change cutoff of 1.5 and p-value of 0.05. Probe set lists were trimmed to represent S. epidermidis and analyzed using unpaired t-test and Benjamini-Hochber multiple-testing correction to generate targeted lists of differential expression. Microarray expression patterns were validated using real-time PCR using three up regulated and two down-regulated genes. The comparison is Mixed-species biofilms vs. S. epidermidis biofilms. However, there are only two biologic replicates per condition [S2 and S3; SC2 and SC3]. It is likely that type II errors are prevalent.     3. A fold change ratio (SC/S) of 2 (or less than 0.5).     Under these filtering conditions, there were 416 interesting genes (there are some genes that are surveyed by more than one probe set). You have file that is called HotGeneList.xlsx. This is an Excel 2007 file. This Excel file is active. You can enter your own filtering criteria. You can enter a p value threshold in the yellow box (cell T9) and or a ratio threshold in the light orange box (cell U9). Then use the filter in cell W10 (Potentially a Hot Gene = 1) to retrieve only genes with a value of “1” in that field, which means the genes meet the filtering criteria.  If you change the filter in either U9 or T9, you must select “all” genes in cell W10 first before re-filtering on “1” in cell W10.   At a p value of 0.05 (and without a ratio filter), we expect by chance alone that 337 genes (out of 6743) will be significantly different (comparing the two groups). Yet we see many more significant genes (n=1059) than can be expected by chance alone a p<0.05. I conclude that there is some real difference between S and SC samples."],"additional_accession":[]},"is_claimable":false,"name":"Transcriptomic analyses of Staphylococcus epidermidis in mixed-species biofilms with Candida albicans","description":"To explain enhanced biofilm formation and increased dissemination of S. epidermidis in mixed-species biofilms, microarrays were used to explore differential gene expression of S. epidermidis in mixed-species biofilms. One sample from single species biofilm (S1) and mixed-species biofilm (SC2) were excluded from analyses for outliers. We observed upregulation (2.7%) and down regulation (6%) of S. epidermidis genes in mixed-species biofilms. Autolysis repressors lrgA and lrgB were down regulated 36-fold and 27-fold respectively and was associated with increased eDNA possibly due to enhanced autolysis in mixed-species biofilms. These data suggest that bacterial autolysis and release of eDNA in the biofilm matrix may be responsible for enhancement and dissemination of mixed-species biofilms of S. epidermidis and C. albicans. Staphylococcal gene expression in mixed-species biofilms with Candida and in single species biofilms of S. epidermidis were analyzed. The experiment was repeated thrice on 3 different days (3 biological replicates each for single species biofilms of S. epidermidis and mixed-species biofilms). Only 2 biological replicates were analyzed and one biological replicate was not analyzed (S1 and SC1 - raw data files are provided on the Series record). Single species biofilms of S. epidermidis (strain 1457) and C. albicans (strain 32354) and mixed-species biofilms were formed on 6-well tissue culture plates. Five ml of organism suspensions (O.D. 0.3, S. epidermidis 107 CFU/ml or C. albicans 105 CFU/ml)  or 2.5 ml each for mixed-species biofilms for 24 hr. RNA was harvested from single species and mixed-species biofilms.","dates":{"release":"2012-02-28T00:00:00Z","modification":"2023-08-20T23:39:40.176Z","creation":"2022-02-07T17:00:25.261Z"},"accession":"E-GEOD-35438","cross_references":{"GEO":["GSE35438"],"EFO":["EFO_0002768"]}}