Genespring 13

RNA isolation and microarray analyses were carried out in accordance with our previously published protocols [14 (link)]. In brief, RNA was isolated using Total Tissue RNA Purification Kit from Norgen-Biotek Corp. (Thorold, ON, Canada) and were quantified using NanoDrop 2000 (Thermo Scientific, Wilmington, DE, U.S.A.). Total RNA was labeled and then hybridized to the Agilent Human SurePrint G3 Human GE 8 × 60k mRNA microarray chip (Agilent Technologies). All microarray experiments were conducted at the Microarray Core Facility (Stem Cell Unit, Department of Anatomy, King Saud University College of Medicine). Data were subsequently normalized and analyzed using GeneSpring 13.0 software (Agilent Technologies). Pathway analyses were conducted using the Single Experiment Pathway analysis feature in Gene Spring 13.0 (Agilent Technologies). Twofold cut-off with P < 0.02 was used. Target prediction was conducted using a built-in feature in Gene Spring 13.0 based on Target Scan database.

The total RNA (150 ng) was labeled using the low input Quick Amp Labeling Kit (Agilent Technologies, Santa Clara, CA, USA), then hybridized to the Agilent SurePrint G3 Human GE 8 × 60 k microarray chip (Agilent Technologies). All microarray experiments were performed at the Microarray Core Facility (Stem Cell Unit, King Saud University College of Medicine, Riyadh, Saudi Arabia). The extracted data were normalized and analyzed using GeneSpring 13.0 software (Agilent Technologies). Pathway analysis was performed using the Single Experiment Pathway analysis feature in GeneSpring 13.0 (Agilent Technologies) as previously described [14 (link)]. Twofold cutoff and a P < 0.05 were used to enrich for significantly changed transcripts.

RNA isolation and gene expression analyses were carried out as described in our previously published manuscripts [44 (link), 45 (link)]. In brief, RNA was isolated using the Total Tissue RNA Purification Kit from Norgen-Biotek Corp. (Thorold, ON, Canada) and was quantified using NanoDrop 2000 (Thermo Scientific, Wilmington, DE, USA). Total RNA was labelled and then hybridized to the Agilent Human SurePrint G3 Human GE 8 × 60 k mRNA microarray chip (Agilent Technologies, Santa Clara, CA, USA). All microarray experiments were conducted at the Microarray Core Facility (Stem Cell Unit, Department of Anatomy, King Saud University College of Medicine). Data were subsequently normalized and analysed using GeneSpring 13.0 software (Agilent Technologies). Pathway analyses were conducted using the Single Experiment Pathway analysis feature in Gene Spring 13.0 (Agilent Technologies). Twofold cut-off with p < 0.02 was used.

RNA isolation, gene and microRNA expression analysis were performed in accordance with our previously published protocols [11 (link)]. In brief, RNA was isolated using Total RNA Purification Kit from Norgen-Biotek Corp. (Thorold, ON, Canada) and were quantified using NanoDrop 2000 (Thermo Scientific, Wilmington, DE, USA). Total RNA was labelled and then hybridized to the Agilent Human SurePrint G3 Human GE 8 × 60 k mRNA microarray chip (Agilent Technologies). All microarray experiments were conducted at the Microarray Core Facility (Stem Cell Unit, College of Medicine, King Saud University). Data were subsequently normalized and analyzed using GeneSpring 13.0 software (Agilent Technologies). Pathway analyses were conducted using the Single Experiment Pathway analysis feature in GeneSpring 13.0 (Agilent Technologies). The Benjamini-Hochberg false discovery rate (FDR) multiple testing correction method [p(corr) < 0.02] was utilized and two fold cut-off were used to enrich for significantly changed transcripts.

RNA concentration and quality were determined with Agilent TapeStation 2200 (Agilent Technologies, Santa Clara, CA, USA). For each sample, 500 ng of total RNA were used to synthesize biotinylated cRNA with Illumina RNA Amplification Kit (Ambion, Austin, TX, USA). Synthesis was carried out according to the manufacturers’ instructions. cRNA concentration and quality were assessed by Agilent TapeStation 2200 (Agilent Technologies). Technical replicates were produced and 750 ng cRNA were hybridized for 18h to MouseWG-6 v2.0 Expression BeadChip (Illumina, San Diego, CA, USA). Hybridized chips were washed and stained with streptavidin conjugated Cy3 (GE Healthcare, Milan, Italy). BeadChips were dried and scanned with an Illumina iScan (Illumina). Gene expression analysis was performed using Gene-Spring 13.1 (Agilent Technologies, Santa Clara, CA, USA). Multichip average (RMA) normalization and principal component analysis (PCA) were performed using Gene-Spring 13.1 (Agilent Technologies, Santa Clara, CA, USA). Fold-change threshold was set at ≥1.5 and statistical significance was set at p ≤ 0.05 by t-test. The Array data are made publicly available upon publication of the paper at https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-7388/, accession number: E-MTAB-7388.

One hundred fifty nanograms of total RNA from day 10 osteoblastic differentiated hBMSCs were labeled using a low input Quick Amp Labeling Kit (Agilent Technologies, Santa Clara, CA, http://www.agilent.com) and hybridized to the Agilent Human SurePrint G3 Human GE 8 × 60 k microarray chip. All microarray experiments were accomplished at the Microarray Core Facility (Stem Cell Unit, Department of Anatomy, King Saud University College of Medicine, Riyadh, Saudi Arabia). The resulted data were normalized and analyzed using GeneSpring 13.0 software (Agilent Technologies). Pathway analysis was performed using GeneSpring 13.0 as described previously [18 (link)]. Twofold cutoff and P(corr) < 0.05 (Benjamini-Hochberg multiple testing corrected) were used to define significantly changed transcripts. Pathway and functional annotation analysis were conducted using the Ingenuity pathway (Ingenuity Systems, http://www.ingenuity.com) [19 (link)]. Differentially expressed genes exhibiting ≥2 FC (fold change) and corrected P value < 0.05 were chosen for analysis. Enriched network categories were algorithmically generated based on their connectivity and ranked according to Z score.