Taqman gene expression assays primers and probes

Total RNA was extracted after homogenization of cells and tissues using RNeasy mini kit (Qiagen Sciences, Inc., Germantown, MD, USA). Total RNA (1 µg) was reversely transcribed with the High Capacity cDNA Reverse Transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The cDNA reaction was diluted to 1:10 for use as template for RT-qPCR. TaqMan Gene Expression Assays primers and probes specific to BAG3 or β-catenin were used for expression analysis and 18S ribosomal primers and probes (Applied Biosystems; Thermo Fisher Scientific, Inc.) were used as internal controls. PCR amplifications were performed in a final reaction volume of 10 µl containing, 5.5 µl of TaqMan Universal PCR Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.), 0.5 µl of the primers and probes mix and 4.5 µg of the cDNA diluted solution. The primers for qPCR are: BAG3-forward: 5′-TGGGAGATCAAGATCGACCC-3′; BAG3-reverse: 5′-GGGCCATTGGCAGAGGATG-3′. All qPCR reactions were carried out in triplicate and repeated at least twice. The ΔCq for mRNA expression was calculated relative to the Cq (quantitation cycle) of 18S ribosomal RNA. Relative mRNA expression was calculated using the formula 2^−ΔΔCq.