Gene specific primer sets

Total RNA was isolated using the Ultraspec RNA isolation system (Biotecx Laboratories). cDNAs were synthesized from total RNA and analysed using gene-specific primer sets (Invitrogen) together with SYBR Green PCR Master mix (PerkinElmer) in an iCycler (Bio-Rad) [26–29 (link)]. Gene expression (relative levels) was calculated after normalization to β-actin using the ΔΔCT method (Bio-Rad).
Real-time quantitative RT-PCR primer sets used are as follows.
PlgR_KTLeft primer5’ ggc att gca acc atc tct tt 3’
Right primer5’ gtt ccg tag ccc agg tca ta 3’
β-actinLeft primer5’ tgg aat cct gtg gca tcc atg aaa c 3’
Right primer5’ taa aac gca gct cag taa cag tcc g 3’
TNFαLeft primer5’ cgt cag ccg att tgc tat ct 3’
Right primer5’ cgg act ccg caa agt cta ag 3’
IL6Left primer5’ gac aac cac ggc ctt ccc ta 3’
Right primer5’ gcc tcc gac ttg tga agt ggt 3’
CCL2/MCP-1Right primer5’ agc acc agc caa ctc tca c 3’
Left primer5’ tct gga ccc att cct tct tg 3’
PPARγ Left primer5’ ctg tcg gtt tca gaa gtg cct 3’
Right primer5’ ccc aaa cct gat ggc att gtg aga ca 3’
AdiponectinLeft primer5’ ctc ctg ctt tgg tcc ctc ca 3’
Right primer5’ gtg cca tct ctg cca tca cg 3’

cDNAs synthesized from total RNA (Ultraspec RNA isolation system; Biotecx Laboratories) were analysed with gene-specific primer sets (Invitrogen) and SYBR Green PCR Master mix (PerkinElmer) in an iCycler (Bio-Rad) [28 (link),29 (link)]. Relative gene expression levels were calculated after normalization to β-actin using the ΔΔCT method (Bio-Rad). Separate control experiments demonstrated that the efficiencies of target and reference (i.e. β-actin) amplifications were equal thus validating the use of ΔΔCT for all of the transcripts that were measured.