Qpcrmix hs sybr lowrox kit

Total RNA was extracted from the isolated glomeruli samples using the “ExtractRNA Reagent” (Evrogen, Moscow, Russia) according to the manufacturer’s protocol. The samples containing 1 μg of RNA were transcribed to cDNA using the random oligodeoxynucleotide primers and the “MMLV RT kit” (Evrogen, Moscow, Russia). PCR amplification was performed using the mixture containing 10 ng of reverse-transcribed product, 0.4 μM of the forward and reverse primers, and the “qPCRmix-HS SYBR + LowROX kit” (Evrogen, Moscow, Russia). The amplified signals were detected using the “Applied Biosystems^® 7500 Real-Time PCR System” (Life Technologies, Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA). The following RT-PCR amplification protocol was used: (I) initial denaturation at 95 °C for 5 min; (II) three-segment amplification and quantification program consisting of 38 cycles: 95 °C for 30 s, 55–56 °C for 10 s, and 72 °C for 30 s; and (III) the ABI Melt Curve program to check for the presence of a single peak and the absence of primer-dimer formation in each reaction containing a template. The primers that were used in the work are presented in Table 2. The expression of the gene encoding 18S rRNA was used as an endogenous control. The relative expression levels of the target genes were calculated by ΔΔC_t method.

Total RNA from macrophages was isolated using the TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. Reverse transcription was performed using 1.5 μg total RNA and random non-amers as primers with RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, USA) according to manufacturer’s protocol. Real-time quantitative PCR was performed using qPCRmix-HS SYBR + LowROX Kit (Evrogen, Moscow, Russia) on the ABI 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Amplifications were performed using the following program: preheating stage at 95°C for 10 min, 40 cycles at 95°C for 15 s, annealing at 61°C for 30 s, and extension at 72°C for 20 s. The following primers were used: IL-6, F: 5′-CTCTGCAAGAGACTTCCATCC, R: 5′-TTCTGCAAGTGCATCATCGT; TNF, F: 5′-TCTGTCTACTGAACTTCGGG, R: 5′-TTGGTGGTTTGCTACGAC; IL-1β, F: 5′-TCAACCAACAAGTGATATTCTCCAT, R: 5′-ACTCCACTTTGCTCTTGACTTCT; and β-actin, F: 5′-GACCTCTATGCCAACACAGT, R: 5′-AGAAAGGGTGTAAAACGCAG. Relative expression of target genes was determined using the ΔΔCt method and normalized to β-actin expression.

Total RNA from macrophages was isolated using the TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. Reverse transcription was carried out using 1.5 mcg total RNA and oligo(dT)₁₈ primers with RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA) according to manufacturer's protocol. Real-time quantitative PCR was performed using qPCRmix-HS SYBR+LowROX Kit (Evrogen, Moscow, Russia) on the ABI 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). RT-PCR analysis was performed as described previously (Korneev et al., 2015 (link)). The following primers were used: IL-6, 5′-CTC TGC AAG AGA CTT CCA TCC, 5′-TTC TGC AAG TGC ATC ATC GT; TNF, 5′-TCT GTC TAC TGA ACT TCG GG, 5′-TTG GTG GTT TGC TAC GAC; IL-1β, 5′-TCA ACC AAC AAG TGA TAT TCT CCA T, 5′-ACT CCA CTT TGC TCT TGA CTT CT; RANTES, 5′-CCC TCA CCA TCA TCC TCA C, 5′-CCT TCG AGT GAC AAA CAC GA; IP-10, 5′-AAG TGC TGC CGT CAT TTT CT, 5′-GTG GCA ATG ATC TCA ACA CG; IRF3, 5′-AAC CGG AAA GAA GTG TTG CG, 5′-GCA CCC AGA TGT ACG AAG TC and β-actin, 5′-GAC CTC TAT GCC AAC ACA GT, 5′-AGA AAG GGT GTA AAA CGC AG.