RNA Extraction and qRT-PCR Analysis

RNA was extracted from RNAi-treated Cont, T2-KD, CRISPR/Cas9 TIMP-2 edited gRNA1, gRNA2, CRISPR control and parental OVCAR5 cells using TRIzol® reagent (Ambion-Life Technologies, Carlsbad, CA, USA) followed by the chloroform: phenol method as described previously [16 (link)]. Five hundred ng of total RNA was reverse transcribed using the high-capacity cDNA Reverse Transcription Kit (Applied Biosystems, CA, USA) and qRT-PCR amplification was performed using the Applied Biosystems ViiA 7 Real-Time PCR (Thermo Fisher Scientific, NSW, Australia) as described previously [16 (link), 26 (link)]. Additional file 1: Table S1 lists the sequences and accession numbers of genes analysed. Data are presented as relative expression normalized to housekeeping gene 18S. The experiments were repeated three times in triplicate.