The miRNeasy Mini Kit (Qiagen) was used for RNA extraction per manufacturer’s instructions. Quality and concentration of total RNA was assessed using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific). For mature miRNA measurements, 10–20 ng of total RNA were reverse transcribed using Taqman microRNA reverse transcription kit (Life Technologies, Foster City, CA) and the product was diluted 1:15 in RNAse-free water. Levels of mature miRNAs, as well as normalizers RNU44 and U6 snRNAs, were assayed in triplicates following quantitative real time polymerase chain reaction with Taqman qRT-PCR assays (Life Technologies) and analyzed using the formula C = 2^Ctgeomean of normalizers / 2^CtmiRNA. Both reverse transcription and qRT-PCR miRNA primers were part of Taqman miRNA assays (Life Technologies). miR-34a expression was normalized to U6 and RNU44 expression to control for the total amount of RNA present in each sample. For mRNA measurements, 300 ng of RNA were reverse transcribed using the VILO cDNA synthesis kit (Invitrogen). The geometric mean of β-actin and 18S rRNA or 18S rRNA alone was used for normalization using the following formula: C = 2^Ctnormalizers / 2^CtmRNA.
