Maxima h minus first strand cdna synthesis kit

Total RNA was extracted from human biopsies and mice tissues using TRIzol Reagent (Thermo Fisher Scientific) and purified RNA was quantified by a Nanodrop spectrophotometer. cDNA was synthesized with the Maxima H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). Real time quantitative-PCR (RT-qPCR) was carried out using the CFX Connect Real-Time PCR Detection System (Bio-Rad, California, United States) with iTaq Universal SYBR Green Supermix (Bio-Rad, California, United States) under the following RT-qPCR cycling parameters: 95°C for 60 s, then 95°C for 15 s and 60°C for 60 s for 40 cycles, 65°C for 5 s. Relative gene expression was standardized to GAPDH and calculated by the 2^−ΔΔCT method. The following primers were used: 5′-TGT​TGC​CAT​CAA​TGA​CCC​CTT-3′ and 5′-CTC​CAC​GAC​GTA​CTC​AGC​G-3′ for human GAPDH; 5′-AGG​GCA​GAA​TCA​TCA​CGA​AGT-3′ and 5′-AGG​GTC​TCG​ATT​GGA​TGG​CA-3′ for human VEGFA; 5′-GAG​ATG​TCC​CTG​GAA​GAA​CAC​A-3′ and 5′-GAG​TGG​GAT​GGG​TGA​TGT​CAG-3′ for human VEGFB; 5′-AGG​TCG​GTG​TGA​ACG​GAT​TTG-3′ and 5′-TGT​AGA​CCA​TGT​AGT​TGA​GGT​CA-3′ for mouse GAPDH; 5′- TAT​TCA​GCG​GAC​TCA​CCA​GC-3′ and 5′- AAC​CAA​CCT​CCT​CAA​ACC​GT-3′ for mouse Vegf.

Nematodes were collected in 1 ml Trizol, and lysed by freeze-thawing. RNA was extracted from lysates with the Qiagen RNeasy Mini Kit (Qiagen), DNAse-treated (ThermoScientific), and used for cDNA synthesis with the Maxima H Minus First Strand cDNA Synthesis Kit (ThermoScientific). qRT-PCR was performed with the KAPA SYBR FAST qPCR Master Mix (KAPA Biosciences) on the LightCycler 480 II real-time thermocycler (Roche Applied Science). Raw values were normalized to each of three control genes (act-1, nhr-23, and ama-1); the average of the normalized values was then used for analysis [75 (link)]. Primer sequences used in qRT-PCR are presented in Additional file 3: Table S3.

RNA was extracted from cultures growing exponentially in 23.43μM fluconazole using the QIAGEN RNeasy^® kit. 1μg of isolate was treated with DNAse and analyzed using an Agilent Bioanalyzer to quantify nucleic acid concentration and verify purity. cDNA synthesis was performed using a combination of oligo-DT and random hexamer primers using the Thermo Scientific™ Maxima™ H Minus First Strand cDNA Synthesis Kit. qPCR on these samples was then performed using a Bioline SensiFAST™ SYBR No-ROX qPCR kit and Ct values were quantified using a CFX machine. cDNA synthesis and qPCR was performed for PDR5 and UBC6 (which acted as loading control).