(This last is significant, because doctors who think patients won't follow their instructions treat and prescribe for them differently).
Instructions play a big part in "Corridor": Mr. Serle, his voice transformed electronically into a rough burble, offers wishes that the dancers enact; all crawl along the floor robotically reciting instructions ("Treat yourself to a tummy tuck") and laughing manically.
Total RNA extracts were prepared with Trizol Reagent (Invitrogen) according to the manufacturer's instructions, treated with DNAse I then reverse transcribed with iScript cDNA synthesis kit (Biorad).
Total RNA was isolated according to the manufacturer's instructions, treated with DNase and immediately reverse transcribed by means of random hexamer primers (Roche) and Superscript II RT (Invitrogen, Life Technologies).
Total RNA was extracted from specimens at 7, 15 and 30 days using TRI Reagent (Sigma, Milan, Italy), following the manufacturer's instructions, treated with DNase (Promega) to exclude DNA contamination and stored at −80°C until the assays.
The following day, cells were transfected with indicated cDNA plasmids using lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions, treated with 10 nM DHT (Wako; dissolved in ethanol) using DMEM supplemented with charcoal:dextran stripped FBS (Gemini Bio-Products), and cultured until analyzed.
After ProteinaseK treatment 500 μl of Trizol LS (Qiagen, Venlo, Limburg) was added to each sample, vortexed for 10 s, and incubated at 25°C for 5 min. Total RNA was recovered from each sample with the miRNeasy kit (Qiagen, Venlo, Limburg) following the manufacturer's instructions, treated with TURBO-DNAfree (Ambion, Carlsbad, CA), and repurified with the miRNeasy kit.
The data are analysed automatically, and a response is provided to the health worker, with specific instructions for treating the child.
After hybridisation with probes, the membrane was washed and blocked following the manufacturer's instructions before treating with a solution of anti-DIG alkaline phosphatase Fab Fragments (Roche).
