One-step Real-time RT-qPCR analysis

The one-step Real-time RT-qPCR reaction was set up using the light cycler 480 RNA master hydrolysis probes kit (Cat# 04991885001, Roche Applied Sciences). Primers for Atoh1 were custom made by IDT as follows: PrimeTime Primer 1: GGG AGA GAA GGA GAC AAA TTC TT, PrimeTime Primer 2: TTC TGC GGG AGG GTA CT, PrimeTime Probe: PRB-/56-FAM/AA AGT CGA G/Zen/A AGTGCA GAG CGT CC/3IABkFQ/ PrimeTime; The primer and probes for the Hes-1 (Hs00172878_m1), Myc (Hs00153408_m1), DKK1 (Hs00183740_m1), RPL13A (03043885_g1) and Axin2 (Hs00610344_m1) were purchased from ThermoFisher.

Probes (250nl/well) and mRNA (500nl/well) were dispensed into the PCR plate by Echo 555 (Labcyte). RT-qPCR Master mix was dispensed by multi-drop Combi nl (Thermo). The final reaction volume was 5ul. The plates were spin-mixed and sealed and run on the Lightcycler 480 (Roche). When working with multiple plates, RNA was converted to cDNA by running all plates through reverse transcription protocol first (3min at 37 °C). Then the plates were stacked on the CRS catalyst (Thermo) for automated feeding onto to the Lightcycler 480 for Real-time PCR reactions.