Stepone plus system thermocycler

RNA was extracted from gingival samples using TRizol LS Reagent (Invitrogen Life Technologies, Carlsbad, CA, United States) and Mini-BeadBeater (BioSpec 3110BX Mini-BeadBeater-1 High Energy Cell Disrupter, Campinas, São Paulo, Brazil) for 20 s, twice. The resulting RNA was treated with desoxyribonuclease (Ambion™ DNase I, Invitrogen Life Technologies, Carlsbad, CA, United States). cDNA was obtained using the SuperScript^TM Vilo^TM Synthesis Kit for RT-PCR (Invitrogen Life Technologies). Quantitative PCR was performed in StepOne Plus System thermocycler (Applied Biosystems, Foster City, CA, United States). Each reaction was performed with 100 ng cDNA using TaqMan™ Gene Expression Assay (Invitrogen by Thermo Fisher Scientific, Vilnius, Lithuania). Commercial Taqman primers and probes (Invitrogen Life Technologies, Carlsbad, CA, United States) comprised Tlr-2 (Mm01213946_g1), Tlr-4 (Mm00445273_m1), Nlrp3 (Mm04210224_m1), Il-1β (Mm00434228_m1), Il-17 (Mm00439619_m1), Tnf-α Mm00607939_s1) and β-actin (Mm00607939_s1). Relative expression of target genes was calculated by the ΔΔCT method, using β-actin as endogenous control (Pfaffl, 2001 (link)), and expressed as fold changes in relation to control group (SHAM).

Twenty frozen fly heads were used to extract RNA using TRIzol reagent with the help of a Direct‐ zol mRNA kit, according to the manufacturer's instructions. cDNA was prepared from 0.4 μg RNA using the qpcrbiosystems kit (PB30.11‐10). Resulting cDNA was used for Quantitative PCR (qPCR) combining it with TaqMan Gene Expression Master Mix (Applied Biosystems) and TaqMan probes (all Thermo Fisher). qPCR was performed on a StepOnePlus system Thermocycler (Applied Biosystems). Each biological experiment was carried out with three independent technical replicates and normalised to actin in each case. Error bars indicate the standard error from mean for at least three different biological replicates. TaqMan probes used for all experiments are as follows: Drosophila ACC (Dm01811991_m1), Drosophila FASN (Dm01821412_m1), Drosophila TACE/ADAM17(Dm02146367_g1), Drosophila actin (Dm02362162_s1), Human FASN (Hs01005624), Human LDLR (Hs00181192_m1) and Human Actin (Hs01060665_g1).

RNA was extracted using Trizol LS Reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) in a cell disrupter (BioSpec 3110BX Mini-BeadBeater-1 High Energy Cell Disrupter, Campinas, SP, Brazil) for 20 s, twice. After deoxyribonuclease (Ambion™ DNase I, Invitrogen Life Technologies) treatment, cDNA was obtained using the SuperScriptTM ViloTM Synthesis Kit for RT-PCR (Invitrogen Life Technologies). Quantitative PCR was performed in StepOne Plus System thermocycler (Applied Biosystems, Foster City, CA, USA) with 100 ng cDNA using TaqMan™ Gene Expression Assay (Invitrogen by Thermo Fisher Scientific, Vilnius, Lithuania). Commercial Taqman primers and probes (Invitrogen Life Technologies, Carlsbad, CA, USA) comprised Tlr-2 (Mm01213946_g1), Tlr-4 (Mm00445273_m1), Il-1β (Mm00434228_m1), Il-10 (Mm01288386_m1), Tnf (Mm00443258_m1), β-actin (Mm00607939_s1), and Gapdh (Mm99999915_g1). Relative expression of target genes was calculated by the ΔΔCT method, using β-actin and Gapdh as endogenous controls [24 (link)], and expressed as fold changes in relation to control group (SHAM).