RNA Extraction and Complementary DNA (cDNA) Synthesis

Cells were thawed on ice, and the RNAlater was removed and discarded. Total RNA was extracted from the cells using the mirVana miRNA Isolation kit (Ambion, Austin, TX, USA). Contaminating genomic DNA in the extracted RNA samples was removed using the TURBO DNA-Free kit (Life Technologies). Total RNA was purified with the RNeasy MinElute Cleanup kit (Qiagen, Hilden, Germany). The quality and concentration of RNA were verified using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and a NanoVue Plus spectrophotometer (GE Healthcare). Single-strand complementary DNA (cDNA) was synthesized from the purified total RNA using the SuperScript III first strand synthesis system (Life Technologies) as following manufacturer’s protocol. The cDNA was purified using QIAquick PCR Purification kit (Qiagen). The purified cDNA was stored at -25°C until qPCR analysis.