Northernmax gly gel prep running buffer

Total RNA from mammalian cells was prepared using the miRNeasy mini kit (Qiagen). One to five micrograms of total RNA or polyA purified RNA (Oligotex, Qiagen) was electrophoresed on a 1% agarose–glyoxal gel, prepared with NorthernMax-Gly gel prep running buffer (Ambion) and transferred to nylon membranes (Hybond XL, GE). Probes were synthesized using a random primer DNA-labelling system (Ambion) with α-32P dATP or T7 MegaScript kit (Ambion) with α-32P UTP. Hybridization was performed at 42 or 68°C overnight in ULTRAHyb hybridization buffer (Ambion) depending on the type of probe used. Following hybridization, membranes were washed, exposed onto an imaging plate and developed using a Typhoon phosphorimager (GE).

Total RNA from mammalian cell lines was extracted with either the miRNEasy mini kit (Qiagen) or the Monarch total RNA miniprep kit (NEB). Depending on the level of expression, 1–4 μg of total RNA was loaded on 1% agarose-glyoxal gel prepared with NorthernMax-Gly gel prep running buffer (Ambion) and transferred to nylon membranes (BrightStar-Plus, Invitrogen). Probes were synthesized using DECAprime II (Invitrogen) with α-³²P dATP, or MEGAscript T3 kit (Invitrogen) with α-³²P UTP. Hybridization was performed overnight at 42 or 68°C in ULTRAHyb hybridization buffer (Ambion). Membranes were then washed and exposed overnight onto imaging plates and imaged on Typhoon phosphorimager (GE). Primers used to amplify both human and mouse Pten probes were (TDO1774) 5′-ACCAGGACCAGAGGAAACCT-3′ and (TDO1775) 5′-GAATGCTGATCTTCATCAAAAGG-3′.