tissue: whole skin biopsy sample type: skin biopsies from pool of 160 patients with skin disorders and healthy donors
Extracted molecule
total RNA
Extraction protocol
Isolation of total RNA from skin excision biopsies was performed using TriReagent (Sigma, St Louis, MO) and the NucleoSpin 96 RNA Kit (Macherey & Nagel, Dueren, Germany). RNA was quantified by photometrical measurement, and the integrity was checked using a 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA).
Label
Cy3
Label protocol
Samples were labeled with FluoroLinkk Cy3/Cy5-dCTP (100 µg total RNA per sample, Amersham Pharmacia Biotech, Freiburg, Germany).
Isolation of total RNA from skin excision biopsies was performed using TriReagent (Sigma, St Louis, MO) and the NucleoSpin 96 RNA Kit (Macherey & Nagel, Dueren, Germany). RNA was quantified by photometrical measurement, and the integrity was checked using a 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA).
Label
Cy5
Label protocol
Samples were labeled with FluoroLinkk Cy3/Cy5-dCTP (100 µg total RNA per sample, Amersham Pharmacia Biotech, Freiburg, Germany).
Hybridization protocol
The fluorescently labeled samples were hybridized overnight to topic-defined PIQOR (TM) Skin 2.0 Microarrays Human Antisense using the GeneTAC hybridisation station (Genomics Solutions).
Scan protocol
Image capture and signal quantification of hybridized PIQOR ™Skin 2.0 microarrays were done with the ScanArrayLite4000 Microarray Scanner (Packard-Biosciences) and ImaGene software Version 5.0 (BioDiscovery, Los Angeles, CA, USA).
Data processing
Signals were analysed with the PIQOR Analyzer software (Miltenyi Biotec, Bergisch Gladbach, Germany). The PIQOR Analyzer allows automated data processing of the raw data text files (_1b = Cy3 channel and _2b = Cy5 channel) derived from the ImaGene software. This includes background subtraction to obtain the net signal intensity, data normalization, and calculation of the Cy5/Cy3 ratios. Subsequently, the mean of the ratios of the four corresponding spots representing the same cDNA was computed. The ratios were normalized using the LOWESS method. As an additional quality filtering step, only genes were taken into account for the calculation of the Cy5/Cy3 ratio that have at least in one channel a signal intensity that was at least 2-fold higher than the mean background