Sisci, D., Aquila, S., Middea, E., Gentile, M., Maggiolini, M., Mastroiami, F., Montanaro, D. and Ando, S.
Notes: MCF-7 cells were incubated for 24 hours in PRF-SFM and then detached and plated on uncoated dishes or dishes coated with 2μg/cm2 poly-L-Lysine (P-Lys) in PBS, 30μg/ml fibronectin (Fn) in PBS or 30μg/ml type IV Collagen (Col) in 10mM acetic acid. The cells plated on uncoated dishes were treated both with and without 10nM estradiol (E2). After 24 hours, total cellular RNA was extracted and reverse transcribed using M-MLV reverse transcriptase. Briefly, reverse transcription was performed on 1μg of total RNA in a final volume of 10μl by incubation at 37°C for 30 min with 200U of M-MLV reverse transcriptase, 0.4μg oligo-dT, 0.5μM dNTP and 24U RNasin® Ribonuclease Inhibitor, followed by heat denaturation for 5 minutes at 95°C. Subsequent PCR analysis was performed on 1μl of the RT product in a final volume of 25μl. Primers were used to amplify the 210bp fragment of PS2 and the 304 bp fragment of cathepsin D. Amplification of 408bp of ribosomal RNA 36B4 was performed as control. The PCR mixture consisted of 1.25U GoTaq® DNA Polymerase, 1X PCR buffer (10mM Tris-HCl, 50mM KCl), 2.5mM MgCl2, 0.2mM each dNTP, 0.6μM of each PS2 primer or cathepsin D primer and 0.2μM of each 36B4 primer. PCR was performed for 20 cycles at 95°C for 1 minute, 59°C for 2 minutes and 72°C for 1 minute. Ten microliters of the PCR products were separated on a 1.2% agarose gel. (3377)