Micro optical plates

Total RNA was isolated (TRI Reagent RNA Isolation system, Sigma #T9424) and DNase I treated (DNAfree TM , Ambion) before reverse transcription (Superscript III First-Strand Synthesis System, Invitrogen) according to the manufacturer's protocol as previously described [24] , except 0.5µl Superscript III was used for each reverse transcription reaction instead of 1µl.
PCR was performed as previously described [25] using GoTaq Green Master Mix (Promega) according to the manufacturer's instructions. Briefly, 1µg cDNA was combined with 2x Master Mix and 10µM primers (sequences shown in Table 1) and performed on a Veriti Thermal Cycler (Applied Biosystems). PCR products were visualized with GelRed Nucleic Acid Stain (Biotium) on a 1.6% agarose gel. qPCR was performed as previously described [10] . Briefly, qPCR analyses were performed on the ABI 7500HT fast block real time PCR system (Applied Biosystems, Foster City, CA, USA) in triplicate in 384-well Micro Optical plates (Applied Biosystems) with the Power SYBR green master mix (Applied Biosystems ) and 200 nM primers (sequences shown in Table 1).
The PCR and qPCR protocol was 95°C for 10 min and 40 cycles of 95°C for 15 s followed by 60°C for 1 min. qPCR relative expression levels were calculated by the comparative cycle threshold method (ΔΔCt), with 18S ribosomal RNA serving as the endogenous control for normalization.