Mesenchymal stem Cells vs. Fibroblasts

PMID Keywords: cell type comparison RNA was prepared from 4 independent samples of cultivated human foreskin fibroblasts and human MSC, respectively using the Nucleospin-II-RNA-Extraction-Kit (Macherey&Nagel). MSC were isolated via their plastic adherence. Briefly, mononuclear cells were isolated from bone marrow by a Ficoll/ Paque Plus (Amersham Biosciences AB, Uppsala, Sweden) gradient separation. RNA samples were quantified by spectrophotometry and RNA integrity was checked by gel electrophoresis (Bioanalyser 2001, Agilent). A RNA pool prepared from equal amounts of all RNA samples was used as common reference for all hybridizations. Linear amplification of RNA was done using a modified protocol of a method described previously (1). Amplified RNA (aRNA) samples were quantified by spectrophotometry and quality was checked by gel electrophoresis (Bioanalyser 2001, Agilent). One microgram of aRNA from each sample was labelled by reverse transcription with Cy5 (individual samples) and Cy3 (common reference) fluorescence, respectively. Labelled samples were then hybridised on PIQOR(TM) Stem Cell Microarrays (Miltenyi Biotec), which contain cDNAs of 942 genes spotted as four-fold replicates on different positions of the array. As a qualitative measurement for the validity of the data and to check for the uniformity of the hybridisation process, the coefficient of variation (cv) of the four ratios for the respective gene was calculated. Hybridisation, scanning and data analysis were performed as described in detail (2). Briefly, image capture and signal quantification of hybridised PIQORTM microarrays were done with the ScanArrayLite4000 Microarray Scanner (PackardBiosciences) and ImaGene software Version 5.0 (BioDiscovery, Los Angeles, CA, USA). Local background was subtracted from the signal to obtain the net signal intensity and the ratio of Cy5/Cy3. Subsequently, the mean of the ratios of 4 corresponding spots representing the same cDNA was computed. The mean ratios were normalised using the Median method. For normalisation only those spots were used for which the fluorescent intensity in one of the two channels was twice the mean background of all unflagged spots. Only genes displaying a net signal intensity 2-fold higher than the mean background were used for further analysis. Two class unpaired significance analysis of microarray (SAM) was performed using the 2-fold change criterion and a false discovery rate of 0,00000%. Only those genes present in all experiments were subjected to SAM.