RNA extraction, cDNA library synthesis and RT-qPCR

2DD cells were grown in 10 cm diameter plates under the previously described treatment conditions. RNA was extracted using TRIzol reagent (Life Technologies, USA, Cat #: 15596026) according to manufacturer’s instructions. RNA concentration was determined using NanoDrop™ 2000 Spectrophotometers (Thermo Fisher Scientific, Wilmington, USA). The quality and integrity of RNA samples was determined by running RNA samples on a 1% agarose gel. RNA samples that showed sharp and clear 28 S and 18 S ribosomal RNA (rRNA) bands were used for cDNA synthesis. Two micrograms of RNA from each treatment were used in cDNA synthesis reactions in combination with random hexamers (50 ng per reaction) and Superscript III (Invitrogen) as per manufacturer’s instructions. The final reaction was diluted to a final volume of 200 μl.

RT-qPCR was conducted using RotorGene qPCR machine (Qiagen). The sequences of the primers used for qPCR were designed by using Primer 3 (version 4.0) software and summarized in Table 1. Ten microliter reactions were set up using 5 μl SYBR Green SuperMix 2x (Quantabio), 2 μl cDNA template, 2 μl H₂O and 1 μl of 3 μM forward and reverse primers. All reactions for each gene were run in triplicate with non-template controls. Results were quantified using the ΔΔCt method against two normalizing genes: SPARC and FKBP10. The fold change values were calculated from ΔΔCt method for antioxidant genes expressed in treatment vs control condition.

Primers for RT-qPCR.