Mx3005p qpcr platform

Total RNA from mammalian cells was extracted using peqGOLD total RNA kit (Peqlab) according to the manufacturer’s directions. RNA was converted to cDNA using Quantitect Reverse Transcription Kit (Qiagen). For quantitative PCR, Brilliant II Sybr green kit (Stratagene/Agilent), including specific MX3005P 96-well semi-skirted plates, was used to analyse samples on the MX3005P qPCR platform (Stratagene/Agilent). Actin was used as a normalising gene in all experiments. The following primers were used for RT-qPCR: actin, F: 5′-CTGGGAGTGGGTGGAGGC-3′, R: 5′-TCAACTGGTCTCAAGTCAGTG-3′. p100, F: 5′-AGCCTGGTAGACACGTACCG-3′, R: 5′-CCGTACGCACTGTCTTCCTT-3′. IL-8, F: 5′-CCAGGAAGAAACCACCGGA-3′, R: 5′-GAAATCAGGAAGGCTGCCAAG-3′. XIAP, F: 5′-CTGTTAAAAGTCATCTTCTCTTGAAA-3′, R: 5′-GACCCTCCCCTTGGACC-3′. HIF-1α, F: 5′-CATAAAGTCTGCAACATGGAAGGT-3′, R: 5′-ATTTGATGGGTGAGGAATGGGTT-3′. A20, F: 5′-ACAGCTTTCCGCATATTGCT-3′, R: 5′-TTGACCAGGACTTGGGACTT-3′. IAP1, F: 5′-AACTCTTGGCCTTTCATTCG-3′, R: 5′-TGTTGTGATGGTGGCTTGAG-3′. IκB-α, F: 5′-AAAGCCAGGTCTCCCTTCAC-3′, R: 5′-CAGCAGCTCACCGAGGAC-3′.
Primer sets for CYLD and DDX3 were obtained from Qiagen.

Total RNA from mammalian cells was extracted using peqGOLD total RNA kit (Peqlab), according to the manufacturer's directions. RNA was converted to cDNA using Quantitect Reverse Transcription Kit (Qiagen). For QPCR, Brilliant II SYBR green kit (Stratagene/Agilent), including specific MX3005P 96 well semi-skirted plates, were used to analyse samples on the Mx3005P QPCR platform (Stratagene/Agilent). Actin was used as a normalizing gene in all experiments. The Primers used for RT–QPCR are shown in Table 2.

RNA was extracted using the peqGOLD total RNA kit (Peqlab), according to the manufacturer's instructions, and reverse transcribed using the QuantiTect Reverse Transcription Kit (Qiagen). For quantitative PCR, Brilliant II Sybr green kit (Statagene/Agilent), including specific MX3005P 96-well semi-skirted plates, was used to analyze samples on the MX3005P qPCR platform (Stratagene/Agilent). Actin was used as a normalizing agent in all experiments. The following primers were used for RT–PCR:

Total RNA, from non-irradiated or 10 Gy irradiated macrophages, was extracted using TriPure Isolation Reagent (Roche), according to manufacturer’s instructions. RNA was converted to cDNA using 150 U of SuperScript™ II Reverse Transcriptase, 1× first strand buffer, 10 mM DTT 0.1 M (Invitrogen), 0.5 mM dNTPs 10 mM (Bioron), 8U of rRNasin (Promega) and RNase/DNase free water (Gibco). To evaluate mRNA expression levels of pro- and anti-inflammatory gene markers, quantitative PCR using Brilliant II Sybr green kit (Stratagene/Agilent Technologies) and specific MX3005P 96-well semi-skirted plates, were performed. Samples were analysed on the MX3005P qPCR platform (Stratagene/Agilent). The following primers, from Invitrogen, were used for RT-qPCR: CXCL8, F: 5′-CCAGGAAGAAACCACCGGA-3′, R: 5′-GAAATCAGGAAGGCTGCCAAG-3′; IL1B, F: 5′-GGCAGGGAACCAGCATC-3′, R: 5′-CCGACCACCACTACAGCAA-3′; TNF F: 5′- GGCTGGAGCTGAGAGATA-3′, R: 5′-CAGCCTTGGCCCTTGAAGA-3′. Primer sets for ACTB (used as a normalizing gene), CXCL12 and CCL2 were obtained from Qiagen, while probes for CD80, CCR7, IL6, CD163, MRC1 and VCAN were from Applied Biosystems.