Quantitative polymerase chain reaction (qPCR) was performed as previously described [22 (link)]. Briefly, TaqMan® Probe-Based Gene Expression Assays supplied by Applied Biosystems, Life Technologies (Carlsbad, CA, USA) were used. To evaluate the effect of SDG treatment on the mRNA expression of antioxidant genes, individual TaqMan® gene expression assays were selected for antioxidant enzymes (heme oxygenase-1 (HO-1), NAD(P)H dehydrogenase, quinone 1 (NQO1), and glutathione S-transferase mu 1 (GSTM1)).
Briefly, cells were pre-treated with SDG (50 μM, 6 h) and irradiated (2 Gy). Total RNA was isolated from using RNeasy Plus Mini Kit (Qiagen, Valencia, CA, USA) and quantified using a NanoDrop 2000 (ThermoFisher Scientific, Waltham, MA, USA). Reverse transcription of RNA to cDNA was then performed on a Veriti® Thermal Cycler using the High Capacity RNA to cDNA kit supplied by Applied Biosystems, Life Technologies (Carlsbad, CA, USA). qPCR was performed using 25 ng of cDNA per reaction well on a StepOnePlus™ Real-Time PCR System (Applied Biosystems, Life Technologies, Carlsbad, CA, USA). Gene expression data was normalized to 18S ribosomal RNA and calibrated to untreated control samples according to the ΔΔCT method as shown previously [22 (link)].
Briefly, cells were pre-treated with SDG (50 μM, 6 h) and irradiated (2 Gy). Total RNA was isolated from using RNeasy Plus Mini Kit (Qiagen, Valencia, CA, USA) and quantified using a NanoDrop 2000 (ThermoFisher Scientific, Waltham, MA, USA). Reverse transcription of RNA to cDNA was then performed on a Veriti® Thermal Cycler using the High Capacity RNA to cDNA kit supplied by Applied Biosystems, Life Technologies (Carlsbad, CA, USA). qPCR was performed using 25 ng of cDNA per reaction well on a StepOnePlus™ Real-Time PCR System (Applied Biosystems, Life Technologies, Carlsbad, CA, USA). Gene expression data was normalized to 18S ribosomal RNA and calibrated to untreated control samples according to the ΔΔCT method as shown previously [22 (link)].
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