Sybr premixex taq 2 tli rnase h plus rox

Cells were transfected, infected or treated [(R)-9b; EPZ6438, TargetMol, Cat#T1788] accordingly. RNA was used for cDNA preparation using High-Capacity cDNA Reverse Transcription Kit (Thermo Scientific Cat#4368814). Resulting cDNA was then analyzed by qPCR using an Applied Biosystems 7900 Real Time PCR System (Thermo Scientific) and SYBR Green PCR Master Mix [SYBR PremixEx Taq II (Tli RNase H Plus) ROX, Clontech Takara, Cat#RR82LR] according to the manufacturers’ instructions. Dissociation curves were generated for each plate to verify the integrity of the primers. The relative expression of RNAs was calculated using the comparative C_t or standard curve method. 18S rRNA or actin were used as internal controls. The primers used are shown in Supplementary Table S1.

NXTAR-N5 oligonucleotide was synthesized by IDT, and the oligonucleotide sequence is provided in Supplementary Table S1 and Fig. 7C (* represents phosphorothioate bond modifications to avoid degradation by exonucleases). Equal numbers of VCaP and 22Rv1 cells were transfected with NXTAR-N5 or globin-derived oligonucleotide using X-tremeGENE360 (Sigma-Aldrich, Cat#08724121001), and the number of live cells were counted 96 h post-transfection using trypan blue exclusion method (Sigma, Cat #T8154). RNA was prepared from these cells, and qRT-PCR [SYBR PremixEx Taq II (Tli RNase H Plus)ROX, Clontech Takara, Cat#RR82LR] for AR and AR-V7 was performed as described earlier (13 (link)).