RNA extraction and reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Total RNA isolation from the cultured Mφs, as well as exudates derived from the pouches of LPS-exposed mice (model described below), was carried out using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The RNA quality was confirmed, through the presence of 28s, 18s and 5s RNA ribosomal subunits in agarose gel electrophoresis and taking into consideration the ratio of OD260/280 to be approximately 2 as well as the ratio of OD260/230 to be approximately 2.2 in all samples.

Reverse transcription was performed from 1.0 μg of purified RNA using the SuperScript Reverse Transcriptase II (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer's instructions. Quantification at the mRNA level was conducted in 96-well polymerase chain reaction (PCR) plates using a Bio-Rad iCycler and the iQ5 Multicolor Real-Time polymerase chain reaction (RT-PCR) detection system (Bio-Rad Laboratories, Inc.). Each reaction contained 1X IQ SYBR-Green Supermix (Bio-Rad Laboratories, Inc.) and 150 nmol/l of each primer. All genes were tested in triplicate. The results were analyzed on the iCycler software. Values were normalized against β-actin. The relative quantification of complementary DNA (cDNA) was performed according to the ΔΔCq method (42). Selected primers are presented in Table SI.

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