Novex minigel system

We ran 8 µg of total RNA per sample on an 8% acrylamide 8M urea gel with a Invitrogen Novex minigel system. Transfer to a nylon membrane was carried out in 0.5X TBE in a Novex XCell II module, followed by UV-crosslinking (1,200J). For detection, we used a combination of 6 oligonucleotide probes targeting FBgn0264479 (5’-gaacatcgcttgcagtgcag, 5’-cgatggatgttgtcggtcgg, 5’-ctctcgttctttgattcttc, 5’-caggatgtgtggtgttccac, 5’-agattggatccttatggttg, 5’-atatgctgacactgcatggt). 30 pmol of oligo mix were radioactively labeled with γ (Guttman and Rinn, 2012 (link))-ATP and PNK. Following phenol-chloroform extraction, the labeled probes were hybridized for 2 hr at 42°C in 40 mL of ULTRAHyb buffer (ThermoFischer). After serial washes with decreasing concentrations of SSC buffer (final stringency 0.5X), the membrane was exposed on Kodak BioMax autoradiography film. After stripping and control re-exposure, a similar protocol was used to detect the 5S rRNA on the same membrane, using a single probe (5’-caacacgcggtgttcccaagccg).

Cell free culture supernatant was analysed for recombinant protein expression with SDS-PAGE electrophoresis system. SDS-PAGE gel electrophoresis of cell free supernatant was carried out using Novex mini gel system (Invitrogen) with Nu-PAGE 10% Bis-Tris gels and MES-SDS running buffer. A known molecular weight (kDa) protein standard marker was loaded in the first well to quantify the unknown weight of the recombinant enzyme samples loaded in the adjacent wells. Finally, the gel was stained with Coomassie Brilliant Blue, destained and then photographed.