Lightcycler detector

MSCs and dTHP-1 were co-cultured as described in Section 2.3. Total RNA was prepared using TRI Reagent (Molecular Research Center, Inc., Cincinnati, OH, USA), following the manufacturer's instructions. Complementary DNAs were prepared from total RNA using AMV (Roche Applied Science, Indianapolis, IN) and random hexamers. Real-time quantitative PCR was performed using LightCycler FastStart DNA Master SYBR Green I and LightCycler detector (both from Roche Applied Science). Assays were conducted in duplicate. Quantitative expression values were extrapolated from standard curves, and normalized to β2-microglobulin (β2M). Specific oligonucleotide primers were: IL-6, 5′-CCCCAGGAGAAGATTCCAAA-3′ (forward primer, F), 5′-CCAGTGATGATTTTCACCAGG-3′ (reverse primer, R); cyclooxygenase-2 (COX-2), 5′-TGAGCATCTACGGTTTGCTG-3′ (F), 5′-TGCTTGTCTGGAACAACTGC-3′ (R); TSG-6, 5′-TCACATTTCAGCCACTGCTC-3′ (F), 5′-AGACCGTGCTTCTCTGTGGT-3′ (R); MCP-1, 5′-CCCCAGTCACCTGCTGTTAT-3′ (F), 5′-TGGAATCCTGAACCCACTTC-3′ (R); RANTES, 5′-CGCTGTCATCCTCATTGCTA-3′ (F), 5′-GAGCACTTGCCACTGGTGTA-3′ (R); β2M, 5′-CCAGCAGAGAATGGAAAGTC-3′ (F), 5′-GATGCTGCTTACATGTCTCG-3′ (R).

Except where specifically indicated in the text, RNA isolation was performed using the RNeasy Mini Kit (Qiagen). cDNA was generated using Superscript III First-Strand Synthesis System (Thermo Fisher Scientifics) using a random hexamer primer. A negative control was generated by replacing reverse transcriptase with water in the cDNA synthesis process to exclude genomic DNA contamination. Messenger RNA (mRNA) expression was measured using quantitative real-time PCR (q-RT-PCR) assays using gene-specific primers (SYBR Green assay) by a LightCycler^® detector (Roche). The relative fold change for each gene was calculated using the ΔΔC_T method as previously described (31 (link)) and the Student's t-test was used to determine statistical significance. Primers used for q-RT-PCR were H4G forward (5′-TGTGATCTGGTACGCCGTG-3′) and reverse (5′-CTGGCGTTTGAGCACGTAGA-3′); H4D forward (5′-GGAAAATGTAATCCHCHATGC-3′) and reverse (5′-CCATAAAGAGTGCGTCCCTG-3′); H4E forward (5′-GTCTACGCGCTGAAGAGACA-3′) and reverse (5′-AGTCGAGATGCTGAGTCGTT-3′); GAPDH forward (5′- CTCCTGCACCACCAACTGCT-3′) and reverse (5′-GGGCCATCCACAGTCTTCTG-3′); and ETS forward (5′-GAACGGTGGTGTGTCGTT-3′) and reverse (5′-GCGTCTCGTCTCGTCTCACT-3′). For quantification of rRNA, total RNA was normalized by GAPDH mRNA and loaded on agarose gels. The 18S and 28S rRNA were quantified using ImageJ software.