One color microarray based gene expression analysis protocol system

Total RNA was isolated from random T1D-MSCs (n = 11) and C-MSCs (n = 10) by the Trizol method (Invitrogen, USA) and purified by RNeasy commercial kit (QIAGEN, USA) according to the manufacturers’ recommendations. RNA integrity was evaluated by microfluidic electrophoresis using Agilent 6000 RNA Nano chips and an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Only RNA samples that were free of proteins and phenol and that featured an RNA Integrity Number (RIN) ≥ 9.0 were used. Random RNA samples of T1D-MSCs and C-MSCs were selected (n = 4 for each group) and the global gene expression was analyzed by the One-color Microarray-Based Gene Expression Analysis Protocol system (Agilent Technologies) on glass slides with four microarrays of 44,000 probes each (4 × 44 k). The preprocess and statistical microarray analyses were performed using algorithms available in the R platform (R Foundation, Vienna, Austria) through the Linear Models for Microarray Data (LIMMA) package [56 (link)]. Heatmaps were generated by the HeatMapViewer module of GenePattern 2.0 software [57 (link)]. Genes with p < 0.05 and fold change (FC) > 2.0 were considered differentially expressed. Microarray data were deposited in the public database ArrayExpress (http://www.ebi.ac.uk/arrayexpress [ArrayExpress:E-MTAB-2976]).

Total RNA was isolated from MSCs from bone marrow of healthy individuals (C-MSCs; n = 4) and MSCs from bone marrow of newly diagnosed T1D patients (T1D-MSCs; n = 4) using Trizol (Invitrogen LifeTechnologies, Carlsbad, CA, USA), according to the manufacturer’s instructions, purified with the RNeasy mini Kit (Qiagen, Valencia, CA, USA), and analyzed by spectrophotometry at 260 and 280 nm (NanoDrop, ND‐1000 UV‐VIS; Thermo Fisher Scientific, Walthman, MA, USA). Global gene expression analyses were performed by the One-color Microarray-Based Gene Expression Analysis Protocol system (Agilent Technologies, Santa Clara, CA, USA) on glass slides with four microarrays of 44,000 probes each (4 × 44 k; Agilent Technologies). The preprocess and statistical microarray analyses were performed using algorithms available from the R platform the Linear Models for Microarray Data (LIMMA, R Foundation, Vienna, Austria) package. The heatmaps were generated by the HeatMapViewer module from GenePattern 2.0 software (Broad Institute, Cambridge, MA, USA). Genes exhibiting P <0.05 and differences in expression of at least 2.0-fold (up or down) were considered statistically significant. Microarray data were deposited in the public database ArrayExpress (http://www.ebi.ac.uk/arrayexpress), access code E-MTAB-2976.