Quantitative RT-PCR

Thank you for your inquiry. To effectively prevent RNA degradation, we took the following precautions: 

We conducted our experiments in an RNAse-free hood. Everything, including the surface, was wiped and disinfected with RNAse AWAY^® (from Molecular BioProducts) and 75% EtOH.  The Eppendorf tubes, tips, and other plastics were certified RNAse, DNAse, and Pyrogen free.

Steps for quantitative RT-PCR:

1. We extracted RNA using Kit from QIAGEN, RNeasy ^® Mini Kit (250) (REF: 74106) and followed the kit instructions. 
2. RNA concentration was measured using nanodrop, with RNA on ice, pipettes and tips remaining RNAse-free.
3. We used the master mix from Applied Biosystems, Fast 1-step master mix (4X) (REF: 4444427). 80 ug RNA was used for each 20 ul system. qPCR was run on QuantStudio 3 from Applied biosystems using the protocol on the master mix instruction. 

(from the instruction)

1. Our primers and probe were ordered from IDT and sequences were listed in the paper.
2. We ran GAPDH on the same plate. Three technical replicates were used for every sample. 
3. We quantified the result by normalizing the Ct value to the GAPDH first, and then to control, using the formula: 2^ΔΔCT .