After MSC-MΦ separation, total RNA was extracted from MSCs and primed MSCs using the RNeasy micro kit (Qiagen) and quantified by use of Nanodrop ND-2000. RNA quality was assayed by the Experion RNA Stdsens analysis kit (Bio-Rad). cDNA was synthesized and amplified using the Ovation PicoSL WTA System (NuGEN). An amount of 25 ng total RNA was reverse transcribed using a primer mix containing both polyT and random sequences for whole-transcriptome coverage, followed by second-strand cDNA synthesis with the Ribo-SPIA technology. The amplified SPIA cDNA was further purified with use of Agencourt RNAClean Beads.
SPIA cDNA was hybridized to a human gene chip (Human Gene 1.1 ST Array Strip; Affymetrix). Array hybridization, washing, and staining were performed as described by the manufacturer (GeneAtlas Hybridization, Wash, and Stain Kit for WT Array Strips; Affymetrix) using the GeneAtlas System. Arrays were scanned on the GeneAtlas Imaging Station (Affymetrix) and analyzed using GeneChip Command Console software (Affymetrix). CEL files were imported into the Partek Genomic Suite (Partek) for normalization and expression comparison. Gene networks representing key genes were identified by using Ingenuity pathways analysis.
SPIA cDNA was hybridized to a human gene chip (Human Gene 1.1 ST Array Strip; Affymetrix). Array hybridization, washing, and staining were performed as described by the manufacturer (GeneAtlas Hybridization, Wash, and Stain Kit for WT Array Strips; Affymetrix) using the GeneAtlas System. Arrays were scanned on the GeneAtlas Imaging Station (Affymetrix) and analyzed using GeneChip Command Console software (Affymetrix). CEL files were imported into the Partek Genomic Suite (Partek) for normalization and expression comparison. Gene networks representing key genes were identified by using Ingenuity pathways analysis.