Agilent 6000 rna nano chips

The total RNA was extracted from approximately 1 x 10⁷ thymocytes, peripheral CD3⁺ T cells or PILs using the mirVana total RNA isolation kit (Ambion, NY, USA) according to the manufacturer´s instructions. The parameters for the quality control of the RNA preparations were previously described [43 (link)]. RNA preparations were confirmed to be free of proteins and phenol by UV spectrophotometry.
The integrity of RNA species was assessed by microfluidic electrophoresis using Agilent 6000 RNA Nanochips and an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Only RNA samples that were free of proteins and phenol and featured an RNA Integrity Number (RIN) ≥ 9.0 were used (data not shown).

Total RNA was extracted using the Illustra RNAspin Isolation Kit (GE Healthcare, Little Chalfont, Buckinghamshire, UK) following manufacturer’s instructions. The quality and concentration of the RNA were checked by measuring the OD 260/280 and OD 260/230. RNA degradation was analyzed by microfluidic electrophoresis using Agilent 6000 RNA Nano chips in an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Samples with an RNA integrity number (RIN) ≥ 9.0 were used.

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]).

T. rubrum cultures (i, ii, and iii) grown for 24 h or co-cultured with keratinocytes were treated with lysis solution (20 mg/mL lysozyme, 0.7 M KCl and 1 M MgSO₄, pH 6.8) for 1 h at 28 °C while shaking (130 rpm) and were collected by centrifugation at 1,000 g for 10 min. The cells were ground with a mortar and pestle and pulverized in liquid nitrogen. Total RNA was extracted using the Illustra RNAspin Mini RNA Isolation Kit (GE Healthcare-Little Chalfont, Buckinghamshire, UK). RNA preparations were confirmed to be free of protein and phenol by UV spectrophotometry. RNA degradation was assessed 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 protein and phenol and had an RNA integrity number (RIN) ≥ 9.0 were used.

After 10 days, the total RNA of each culture was extracted with mirVana total RNA isolation kit® (Ambion, NY, USA), according to the manufacturer's instruction. UV spectrophotometry confirmed that the RNA preparations were free of proteins and phenol. RNA degradation was assessed by microfluidic electrophoresis with Agilent 6000 RNA Nano chips, conducted on an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Only RNA samples free of proteins and phenol and featuring an RNA Integrity Number (RIN) ≥ 9.0 were used.