RNA was extracted using the RNeasy Mini kit (Qiagen) according to the manufacturer’s instructions, and on-column DNase digestion was performed using the RNase-Free DNase Set (Qiagen). The RNA was quantified by spectrophotometric analysis and the quality of RNA was routinely checked by measurement of OD (260/280 and 260/230 ratio) and by gel electrophoresis. cDNA synthesis was performed by reverse transcription of RNA using the Reverse Transcription System (Promega). Real-time quantitative PCR was performed using the iCycler iQ Real-Time Detection System (Bio-Rad Laboratories) as described before (17). Human TNF-α primers were obtained from Maxim Biotech and human 18S primers (Forward 5′GTA ACC CGT TGA ACC CCA TT 3′; Reverse 5′ CCA TCC AAT CGG TAG TAG CG 3′) and IRAK-M primers (Forward 5′ AGA GCA GCT GTT CCT CCA AA 3′; Reverse 5′ AAT TGA GCG TGG ATT TGG TC 3′) were synthesized by IDT.
