96 well qpcr plate

Total RNA extraction from different samples was done using the method previously described [41 (link)] and quantified using a UV spectrophotometer. The RT-PCR reactions were performed for selected genes (Table 1) according to the manufacturer’s instructions using 2 × MESAGREEN qPCR MasterMix Plus for the SYBR 258 Assay (Eurogentech, Liège, Belgium) in a 96-well qPCR plate (Sarstedt, Nümbrecht, Germany), and the Mx3005P^TM sequence detection system (Agilent Technologies, Santa Clara, CA, USA). A quantitative analysis was made based on the cycle threshold (Ct) value for each well and calculated using MxPro software (Agilent Technologies, Santa Clara, CA, USA). The results were normalized using the mean Ct of the three housekeeping genes (Ct HKG) (Table 1) and the data are represented as dCt or as fold differences using the 2^−ddCt method, where dCt = Ct target gene − Ct HKG.

The SuperscriptTM II Transcriptase Reverse Kit is used for RT (Invitrogen, Carlsbad, CA, USA) GB. Reverse transcription was performed using 1 µg of total RNA. The RT-PCR reactions were performed, for selected genes (Table 1), according to the manufacturer’s instructions using 2× MESA GREEN qPCR MasterMix Plus for SYBR 258 Assay (Eurogentech, Liège, Belgique) 96-well qPCR plate (Sarstedt, Nümbrecht, Germany), optical seal (Dutcher, Brumath, France), and the Mx3005PTM sequence detection system (Agilent technologies, Santa Clara, CA, USA). In each reaction, 10 ng of reverse transcripted RNA (based on initial RNA concentration) was used. All primers were used at 400 nM in a 20 µL reaction. Quantitative analysis was made based on the cycle threshold (Ct) value for each well and calculated using the MxPro software (Agilent technologies, Santa Clara, CA, USA). The results are normalized by three housekeeping (HKG) genes: 18S, GAPDH, and HPRT (Table 1), and data are represented as fold differences by the 2^−ΔΔCt method, where ΔCt = Ct target gene—Ct HKG.

Isolation of total RNA was performed using RNeasy Mini Kit (Qiagen) according to manufacturers’ instructions. After treatment, cells were collected, washed with 1xPBS and lysed in Buffer RLT (containing 1% ß-mercaptoethanol). All following steps were conducted as described in the manufacturers’ protocol. RNA concentration and quality were determined using a Nanodrop 2200 (ThermoFisher). Only samples showing a 260/280 nm ratio between 1.8 and 2.1 were selected for cDNA transcription, which was performed with the Omniscript RT Kit (Qiagen) and random hexamers (Life Technologies). Quantitative PCR (qPCR) analysis was done using TaqMan® primers and a StepOnePlus System (Applied Biosystems). Briefly, for each well of the 96-well qPCR plate (Sarstedt), 10 µl of TaqMan™ Universal PCR Master Mix (ThermoFisher) were mixed with 10 ng cDNA and 1 µl of the appropriate primer (ThermoFisher). All measurements were performed using three technical replicates. Relative quantification (RQ) of gene expression were determined using the 2^−ΔΔCt method. Primer IDs: DDIT3 (Hs00358796_g1), GAPDH (Hs02758991_g1); DEGS1 (Hs00186447_m1); SK1 (Hs00184211_m1); SK2 (Hs01016543_g1). Expression of GAPDH, used as a reference gene, was similar in untreated cells under both 21% and 3% oxygen conditions (Ct values of 20,743 and 20,749 respectively).