LX2 cells were incubated with complete DMEM with or without 2.5 mM VPA respectively for 24 hr. Total RNAs were extracted from VPA treated or untreated LX2 cells using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. miRNA microarray experiments using TaqMan™ Array Human MicroRNA A+B Cards Set v3.0 (Applied Biosystems, Foster City, CA) were performed according to the standard protocol, each with a biological replica. Raw Ct values of the miRNA microarray for VPA treated LX2 cells and untreated control LX2 cells were normalized to an endogenous control U6 snRNA18 (link).
The RNAs from VPA treated or untreated LX2 cells were reverse-transcripted with Thermoscript RT-PCR system (Invitrogen) or NCode miRNA First-Strand cDNA Synthesis kits (Invitrogen). For quantitative detection of mRNAs and mature miRNAs, the templates and specific gene primers (TableS1 ) were mixed with SYBR Premix ExTaq (Takara, Tokyo, Japan), and q-PCR was performed using Rotor-Gene Q thermocycler (QIAGEN, Germany). The relative expressions of mRNAs and miRNAs were calculated as 2-ΔCt values, normalized to housekeeping gene β-actin or U6snRNA respectively.
The RNAs from VPA treated or untreated LX2 cells were reverse-transcripted with Thermoscript RT-PCR system (Invitrogen) or NCode miRNA First-Strand cDNA Synthesis kits (Invitrogen). For quantitative detection of mRNAs and mature miRNAs, the templates and specific gene primers (Table
