Quantitative RT-PCR Analysis of Gene Expression

Total RNA was isolated (SV RNA Isolation kit, Promega, Madison, WI, USA) and qRT-PCR was performed on a Cepheid SmartCycler II (Cepheid, Sunnyvale, CA, USA) using a Superscript III Platinum SYBR Green One-Step qRT-PCR kit (Invitrogen, Carlsbad, CA, USA), as per our laboratory [34 (link), 39 (link), 41 –43 (link)]. Product specificity was confirmed by melting curve analysis and visualization of a single, appropriately sized band on a 2% agarose gel. Gene expression levels were quantified using a cDNA standard curve and data was normalized to RPS3, a housekeeping gene that is unaffected by the experimental manipulations. Data is expressed as fold change versus sham.