NCBI Logo
GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
          Go
Sample GSM1489482 Query DataSets for GSM1489482
Status Public on Oct 01, 2014
Title IL-17A 2
Sample type RNA
 
Source name monocyte-derived macrophages
Organism Homo sapiens
Characteristics cell type: monocyte-derived macrophages
stimulation status: stimulated
Treatment protocol For macrophage polarization, monocyte derived macrophages were stimulated with 10ng/mL IL-17A for 18h. Unstimulated macrophages served as control. After 18h RNA was isolated from macrophages using columns including a DNAse-step followed by reverse transcription. RNA was labeled and hybridized to Illumina expression profiling for whole genome BeadChipSentrix array (kindly provided by Hudler, German Cancer Research Center). For blood withdrawal, patients gave written consent, and the study was approved by the local institutional ethics committee. To investigate the impact of IL-17A on macrophage differentiation, isolated monocytes were incubated either with 100ng/mL rhM-CSF or with 10ng/mL IL-17A or both for 6 days.
Growth protocol Human monocyte-derived macrophages were generated as described previously (Gleissner at al., Arterioscler Thromb Vasc Biol 2008)
Extracted molecule total RNA
Extraction protocol After 6 days for macrophage differentiation, RNA was isolated from macrophages using columns including a DNAse-step followed by reverse transcription (all reagents from Qiagen).
Label biotin
Label protocol The laboratory work was done in the Genomics and Proteomics Core Facility at the German Cancer Research Center, Heidelberg, Germany (DKFZ). Biotin-labeled cRNA samples for hybridization on Illumina Mouse Sentrix-6 BeadChip arrays (Illumina, Inc.) were prepared according to Illumina's recommended sample labeling procedure based on the modified Eberwine protocol (Eberwine et al., 1992). In brief, 100 ng total RNA was used for complementary DNA (cDNA) synthesis, followed by an amplification/labeling step (in vitro transcription) to synthesize biotin-labeled cRNA according to the Illumina® Total Prep™ RNA Amplification Kit (Life Technologies). Biotin-16-UTP was purchased from Roche Applied Science, Penzberg, Germany. The cRNA was column purified according to TotalPrep RNA Amplification Kit, and eluted in 60 µl of water. Quality of cRNA was controlled using the RNA Nano Chip Assay on an Agilent 2100 Bioanalyzer and spectrophotometrically quantified (NanoDrop).
 
Hybridization protocol Hybridization is performed at 58°C, in GEX-HCB buffer (Illumina Inc.) at a concentration of 100 ng cRNA/µl, unsealed in a wet chamber for 20h. Spike-in controls for low, medium and highly abundant RNAs were added, as well as mismatch control and biotinylation control oligonucleotides. Microarrays were washed once in High Temp Wash buffer (Illumina Inc.) at 55°C and then twice in E1BC buffer (Illumina Inc.) at room temperature for 5 minutes (in between washed with ethanol at room temperature). After blocking for 5 min in 4 ml of 1% (wt/vol) Blocker Casein in phosphate buffered saline Hammarsten grade (Pierce Biotechnology, Inc., Rockford, IL), array signals are developed by a 10-min incubation in 2 ml of 1 µg/ml Cy3-streptavidin (Amersham Biosciences, Buckinghamshire, UK) solution and 1% blocking solution. After a final wash in E1BC, the arrays are dried and scanned.
Scan protocol Microarray scanning was done using an iScan array scanner. Data extraction was done for all beads individually, and outliers are removed when > 2.5 MAD (median absolute deviation). All remaining data points are used for the calculation of the mean average signal for a given probe, and standard deviation for each probe was calculated.
Data processing The analysis is done with R on the bead-level data which you can find in the raw data produced by the scanner. All analyses are done using the following mechanisms: 1) outlier removal (see below). 2) average values over beads are means (not median). 3) statistical tests are t-tests over all beads (e.g for one probeID over all beads of all samples of a group) in original scale (not log2 scale). Calculation of GeneView data: The calculations are also done on the set of beads which belong to a given Gene Symbol (annotation column Symbol). Outlier removal on bead level: Outliers are removed by taking only beads with an expression value > 20 before using the 2.5 MADs rule. The ProbeIDs expression value is than the mean of the remaining beads. The remaining bead level lists of different length for each sample are randomly filled with NAs (NA=not available) and build the bead level matrix on which quantile normalization is performed. MAD rule: mad = median absolute deviation, x = one bead level expression value of some specific probeID, Than: outliers are those x where abs((x-median)/mad) > 2.5 The analysis is done with R on the bead-level data which you can find in the raw data produced by the scanner. All analyses are done using the following mechanisms: 1) outlier removal (see below). 2) average values over beads are means (not median). 3) statistical tests are t-tests over all beads (e.g for one probeID over all beads of all samples of a group) in original scale (not log2 scale). Calculation of GeneView data: The calculations are also done on the set of beads which belong to a given Gene Symbol (annotation column Symbol). Outlier removal on bead level: Outliers are removed by taking only beads with an expression value > 20 before using the 2.5 MADs rule. The ProbeIDs expression value is than the mean of the remaining beads. The remaining bead level lists of different length for each sample are randomly filled with NAs (NA=not available) and build the bead level matrix on which quantile normalization is performed. MAD rule: mad = median absolute deviation, x = one bead level expression value of some specific probeID, Than: outliers are those x where abs((x-median)/mad) > 2.5 The analysis is done with R on the bead-level data which you can find in the raw data produced by the scanner. All analyses are done using the following mechanisms: 1) outlier removal (see below). 2) average values over beads are means (not median). 3) statistical tests are t-tests over all beads (e.g for one probeID over all beads of all samples of a group) in original scale (not log2 scale). Calculation of GeneView data: The calculations are also done on the set of beads which belong to a given Gene Symbol (annotation column Symbol). Outlier removal on bead level: Outliers are removed by taking only beads with an expression value > 20 before using the 2.5 MADs rule. The ProbeIDs expression value is than the mean of the remaining beads. The remaining bead level lists of different length for each sample are randomly filled with NAs (NA=not available) and build the bead level matrix on which quantile normalization is performed. MAD rule: mad = median absolute deviation, x = one bead level expression value of some specific probeID, Than: outliers are those x where abs((x-median)/mad) > 2.5
As test for significance the student’s t-test is used on the bead expression values of the two groups of interest. In the case of significance of expression against background we tested for greater than all negative beads for this sample and in the case of comparing separate groups we tested for inequality of the means of the groups. In both cases Benjamini-Hochberg correction was applied to the complete set of p-values of all ProbeIDs on the chip. The average expression value is calculated as mean of the measured expressions of beads together with the standard deviation of the beads.
 
Submission date Aug 27, 2014
Last update date Oct 01, 2014
Contact name Christian Albert Gleissner
E-mail(s) gleissner.christian@rottalinnkliniken.de
Phone +49-8721-9837302
Organization name Rottal-Inn Kliniken KU
Department Cardiology
Lab Gleissner
Street address Simonsöder Allee 20
City Eggenfelden
ZIP/Postal code 84307
Country Germany
 
Platform ID GPL6884
Series (1)
GSE60824 IL-17A influences essential functions of the monocyte / macrophage lineage and is involved in advanced murine and human atherosclerosis

Supplementary file Size Download File type/resource
GSM1489482_IL17A_3_5388913006_F_raw.txt.gz 322.9 Kb (ftp)(http) TXT
GSM1489482_IL17A_3_5388913006_F_raw_annot.txt.gz 9.0 Mb (ftp)(http) TXT
Raw data provided as supplementary file
Processed data are available on Series record

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap