Perfectpure rna tissue kit

Before each assay, cell pellets from PCa cell lines (LAPC4, LNCaP, PC3, DU145, 22RV1, and C4–2) were thawed on ice and resuspended in 300 μL of lysis buffer; 50 mM Tris-HCl at pH 7.5; 150 mM NaCl; 1% Triton X-100; DNase I (Roche); protease inhibitor cocktail (Promega); and NaF and incubated at RT for 10 min with gentle rotation. The cell suspension was passed through a 27-gauge needle 10 times, and the lysate was centrifuged at 14000g for 5 min at 4 °C. For the mouse xenograft study, 12 pairs of each HS and CR human prostate tissue were prepared. LuCaP patient-derived xenografts were grown subcutaneously in intact (HS lines) and castrated (CR lines) SCID male mice.^{29 (link)} RNA was isolated from the tissues using a Perfect Pure RNA Tissue Kit (5-Prim) in the presence of DNase I. cDNA was synthesized with qScript cDNA superMix (Quanta Biosciences) using 1 μg of total RNA with both Oligo(dT)’s and Random Haxamer Primer present. To further eliminate gDNA contamination, the amplification of cDNA is achieved by designing exon-primed intron crossing primers, in which the forward and reverse primers flank at different exons. qPCR was performed to measure gene expression, and GAPDH was used as a housekeeping gene.