Plasmid dna maxi prep kits

Glutathione S-transferase P1 expression was stably knocked-down in A2780 and expression significantly reduced in A2780DPP cells by RNA interference using Mission shRNA plasmids (Sigma-Aldrich, Gillingham, UK). Five unique GSTP1-specific shRNA plasmids (clones TRCN0000083773, TRCN0000083774, TRCN0000083775, TRCN0000083776 and TRCN0000083777) and a negative control plasmid (empty vector control, SHC001) were purchased as glycerol stocks and plasmid DNA extracted using plasmid DNA maxi prep kits (Qiagen, Manchester, UK) according to the manufacturer's instructions. A2780 and A2780DPP cells (2.5 × 10⁵ cells per well in six-well plates) were transfected with each plasmid using lipofectamine (Life Technologies, Paisley, UK), and shRNA-containing cells selected with puromycin. Individual cell colonies were picked using cloning cylinders, harvested for mRNA analysis (A2780DPP cells) or expanded to 75 cm² tissue culture flasks and harvested for mRNA and protein analysis (A2780 cells). GSTP1 knockdown in A2780 cells was confirmed by qRT–PCR analysis and western blotting, and by qRT–PCR analysis in A2780DPP cells. Cell growth rates were compared by plating 1 × 10⁵ cells from each cell line in individual wells of six-well dishes (day 0). Cells were harvested daily by trypsinisation (days 2–10) and counted using a haemocytometer.