Block it rnai topo transcription kit

Plasmid DNAs were transfected into HeLa cells using X-tremeGENE 9 DNA Transfection Reagents according to the manufacturer's instructions (Roche Applied Science), For knockdown of the SNARE protein, a pool of siRNAs for each SNARE was prepared using the cDNA region shown in supplementary material Table S2 as a template with a BLOCK-iT RNAi TOPO transcription kit (Invitrogen) and a PowerCut Dicer kit (Thermo Scientific). Knockdown was performed as described previously (Takahashi et al., 2011 (link)). Briefly, HeLa cells were transfected with the siRNA pool using Lipofectamine 2000 (Invitrogen) and incubated for 24 h. The cells were then subcultured for 24 h, re-transfected with siRNAs and incubated for further 24 h. The cells were then subcultured, incubated for 48 h, and subjected to following analyses.

For in vitro ER knockdown assessment, RNA was isolated from AsPC-1 and L3.6pl cells using a RNeasy™ Mini Kit (Qiagen; Leipzig, Germany). Reverse transcription was carried out using a SuperScript™ III One-Step RT-PCR System with Platinum™ Taq High Fidelity (Thermo Fisher Scientific, Bremen, Germany) according to the manufacturer’s instructions. ERβsi forward and ERβsi reverse primers were developed in such a way that they amplified 586 bp or 531 bp, respectively. ERαsi: for 5’- GTATGATCCTACCAGACCCTTCAGTGA-3’; rev 5’-AGATGCTCCATGCCTTTGTTAC TC-3’. ERβsi: for 5’-GCCGCCCCATGTGCTGAT-3’; rev 5’-ATGGATTGCTGCTGGGAG GAGATG-3’. siRNAs were generated using BLOCK-iT™ RNAi TOPO Transcription Kits and BLOCK-iT™ Dicer RNAi Kits (both Thermo Fisher Scientific) according to the manufacturer’s instructions. Lipofectamine™ 2000 (Thermo Fisher Scientific) was used for the transfection of L3.6pl cells with the designed siRNAs. Control cells were transfected with siRNAs specific for the lacZ reporter gene, which was generated in the same way, using primers and templates supplied with the kit.