Bioline cdna synthesis kit

qRT-PCR was performed according to the protocol described previously^{2 (link)}. Briefly, cells were first lysed using TRIzol (Invitrogen). Pure RNA was then isolated using the ISOLATE II RNA Mini Kit from Bioline (Alexandria). For tissue samples, snap-frozen tissues were first homogenized using beads (Lysing Matrix D Bulk; MP Biomedicals) in TRIzol. RNA was then isolated as per manufacturer’s instructions using the ISOLATE II RNA Mini Kit from Bioline (Alexandria). 1 μg of RNA was then used to synthesize cDNA, using the Bioline cDNA synthesis kit containing oligo (dT) and random hexamers. All cDNA was then diluted in a 1:10 ratio to perform qRT-PCR.
2.5 μL of diluted cDNA, 0.75 μL of desired primer (2 μM), 3.75 μL of SYBR green (SensiFAST SYBR Lo-ROX kit; Bioline), and 0.5 μL of DNase and RNase free water were mixed and measured on a Real-Time PCR System (Applied Biosystems ViiA 7; Life Technologies Corporation) for 40 cycles. The resulting Ct values were then analysed using the ViiA 7 software (Life Technologies Corporation). The relative expression of target genes was determined by the ΔΔCt method and normalized to housekeeping gene GAPDH or Ywhaz and expressed as a fold difference to the mean of the relevant control samples. See tables S1 (human) and S2 (mouse) for details on primers used in this study.

Total RNA was isolated using TRIzol reagent (Invitrogen) and Aurum Total RNA Isolation Mini Kit (Biorad), following manufacturer’s instructions for processing animal tissue. cDNA was synthesized using Bioline cDNA synthesis kit (Bioline). 10 ng cDNA was used for quantitative PCR in a total volume of 10 uL with LightCycler 480 SYBR Green I Master Mix (Roche) with appropriate primers, on a LightCycler 480 (Roche). All reactions were performed in triplicate. Mouse-specific primers used are as follows: Hprt forward 5’-AGTGTTGGATACAGGCCAGAC-3’ reverse 5’-CGTGATTCAAATCCCTGAAGT-3’, Gapdh forward 5’-TGAAGCAGGCATCTGAGGG-3’ reverse 5’-CGAAGGTGGAAGAGTGGGAG-3’, CCL5 forward 5’-CGTCAAGGAGTATTTCTACAC-3’ reverse 5’-GGTCAGAATCAAGAAACCCT -3’, CCL2 forward 5’-TTAAAAACCTGGATCGGAACCAA-3’ reverse 5’-GCATTAGCTTCAGATTTACGGGT-3’, proIL-1β forward 5’-TGGGCCTCAAAG GAA AGA-3’ reverse 5’-GGTGCTGATGTACCA GTT-3’, proIL-18 forward 5’-CAGGCCTGACATCTTCTGCAA-3’ reverse 5’-TCTGACATGGCAGCCATTGT-3’, IL-6 forward 5’-GAGGATACCACTCCCAACAGACC-3’ reverse 5’-AAGTGCATCATCGTTGTTCATACA-3’, TNF forward 5’-ACCCTGGTATGAGCCCATATAC-3’ reverse 5’- ACACCCATTCCCTTCACAGAG-3’.

The expressions of IL-1β, IL-6, TNF-α, Bax, Bcl2, caspase-3, SIRT1, Nrf2, and HO-1 were determined using RT-PCR. In brief, RNA was obtained using the QIAzol reagent (Qiagen, Hilden, Germany) according to the kit’s instructions. The RNA concentration was estimated using the NanoDrop 2000 (ThermoScientific, Waltham, MA, USA). Reverse transcription of RNA samples (≈1 µg) was performed using the Bioline cDNA synthesis kit (Bioline, Taunton, MA, USA). cDNA replication was carried out using RT-PCR equipment (Pikoreal 96, ThermoScientific, USA). The amplification process was composed of a total volume mixture (20 µL): HERA SYBR green PCR Master Mix (10 µL, Willowfort, West Midlands, UK), cDNA template (2 µL), 2 µL of each gene primer (10 pmol/µL), and nuclease-free water (6 µL). The process was carried out according to the following conditions: 95 °C for 2 min, followed by 40 cycles of 95 °C for 10 s, and 60 °C for 30 s. The studied genes primers were designed utilizing Primer3Plus software, and their specificity was assigned utilizing the Primer-BLAST program. Primer sets synthesis was carried out using Vivantis. GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) was used as a control gene (Table 1). Relative gene expression levels were represented as ∆Ct = Ct target gene − Ct control gene; the fold change in gene expression was calculated according to the 2^−∆∆CT method.