Fig. 2

Analysis of PTEN gene expression by semiquantitative end-point RT-PCR after miR-21 silencing. Total RNA and cDNA synthesis were obtained from 1 × 10⁵ SiHa cells (HPV16+) per well in a six-well plate containing DMEM at 37 °C with 5 % CO₂ after 48 h transfection with pSIMIR21 plasmid. Panel a Analysis of PTEN gene expression by semiquantitative end-point RT-PCR in SiHa cells non transfected (NT, lane 1) or transfected with pSilencer1.0-U6 plasmid or pSilencer2.0-U6 plasmid (empty vectors) or with pSIMIR21 plasmid (lanes 2 to 4). HaCat cell line was used as positive control (H, lane 5). PCR reaction without cDNA corresponds to reaction negative control (C-, lane 6). PCR amplification products were separated by electrophoresis in 1 % agarose gel. The DNA 100 bp ladder was used as molecular weight. Panel b PCR product bands were digitalized and analyzed by densitometer and data were analyzed by mRNA PTEN/mRNA GADPH ratio in relative expression units