Quantigene plex magnetic separation assay kit

For quantification of mRNA levels of Dnm1l and actb in muscle, we used the QuantiGene Plex Magnetic Separation Assay kit (Affymetrix, Santa Clara, CA, USA) following the manufacturer’s instructions and as previously described [32 ]. The signal was detected with a Luminex instrument (Bio-Rad, Milan, Italy). For each sample, the average signal (MFI) for Dnm1l and actb were determined and, after average background signal subtraction, Dnm1l signal was normalized to the housekeeping gene signal.

The QuantiGene Plex Magnetic Separation Assay kit was used (Affymetrix, Santa Clara, CA, USA). All experiments were performed according to manufacturer’s instructions. Briefly, 500 ng of RNA was added to separate wells of a 96-well hybridization plate containing magnetic capture beads and QG_2.0 probe sets. The plate was incubated for 22 h at 55 °C and agitated at 600 rpm to maintain the suspension. Beads were sequentially hybridized (1 h, 50 °C at 600 rpm) with preamplifier probe, the amplifier probe, the label probe and phycoerythrin-conjugated streptavidin, with 3 washes in washing buffer in between, followed by a final 30-min incubation at 37 °C with developing solution. Bead discrimination and signal detection were performed on a Luminex instrument (Bio-Rad, Milan, Italy). For each sample the average signal (MFI) for Ptch1, Smo, Gli1 and actb were determined and, after average background signal subtraction, each gene signal (background subtracted) was divided by the normalization gene signal (background subtracted): actb. Data are shown as mean (± SEM) fold change over control.