RNA was extracted using the EZNA. Total RNA Kit II (Omega Bio-Tek, Norcross, GA, USA) and cDNA was prepared with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Grand Island, NY, USA). The cDNA was amplified to quantify steady-state gene expression by qPCR using TaqMan Universal PCR Master Mix and Prism 7900 thermocycler (Applied Biosystems, Foster City, CA, USA). Using the comparative threshold cycle method (GAPDH used to normalize target gene expression), differences in cDNA levels were determined by comparing 2−ΔΔCts, Ct = cycle threshold (39 (link)).
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