Custom taqman microrna assay

Total RNA was extracted from samples using TRIzol reagent (Life technologies) and reverse transcription was performed using the iScript cDNA Synthesis Kit (Bio-Rad). Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed to detect mRNA expression using Taqman Universal Mix II (Applied Biosystems) with Taqman probes (Applied Biosystems, Mm01731290_g1 for p53, Mm00432448_m1 for Cdkn1a, Mm00449846_m1 for Phlda3, Mm01281680_m1 for Lmo2, Mm01342805_m1 for Hes1, Mm99999915_g1 for Gapdh). Gapdh was used as an internal control to correct for the concentration of RNA in different samples. Expression of miR-30-based p53.1224 shRNA was detected using a Custom TaqMan MicroRNA Assay (Applied Biosystems). A reverse transcription primer and a TaqMan probe were designed to specifically detect p53.1224 siRNA^{56 (link)}. In vitro reverse transcription was performed using TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems). SnoRNA 202 was used as an internal control to correct for the concentration of RNA in different samples. Each experiment was performed with three replicates from each sample, and the results were averaged.