Genescreen plus

Total RNA was isolated from MEF or HEK 293 cells using Trizol (Life technologies) 48 hrs after transfection. For C2C12 cells, RNA was Trizol extracted after each day of differentiation for 5 days. Expression of miR-133a, a marker of muscle differentiation^{44 (link)}, was monitored to validate the differentiation to myotubes.
For small RNA northerns, ten micrograms of total RNA was electrophoresed on 15% (w/v) acrylamide/7M urea gel and transferred onto a Gene screen Plus (Perkin Elmer) membrane. Hybridization was done overnight in Perfect Hyb Plus (Sigma) buffer at 65°C using LNA probes for miR-151-5p and miR-124 (Exiqon) or at 35°C with an oligonucleotide probe (IDT).

Genomic DNA was extracted from the WT and FVYL mutant hTERT transfected fibroblasts using a QIAamp DNA-mini Kit (Qiagen). 10 μg of Genomic DNA from each sample was digested overnight at 37° C using 20 U of MboI (NEB) and 20 U of AluI (Invitrogen). Digested DNA was precipitated in 10 mM NaOAc, 1 mM MgCl₂, and 75% ethanol overnight at −20° C, then pelleted and resuspended in DNase free water. 3 μg of each sample were run on a 0.5% agarose gel in 1X Tris acetate EDTA (TAE) buffer for 4.5 hours at 120 V. The gel was sequentially washed with 0.25 M HCl, denaturing buffer (0.5 M NaOH, 1.5 M NaCl), and neutralization buffer (0.5 M Tris-HCl, 3 M NaCl, pH 7.5), then blotted onto hybridization transfer membrane (Genescreen Plus^™, Perkin Elmer Health Sciences). DNA was cross-linked to the membrane using a UV Stratalinker 1800 (Stratagene), and hybridized with 0.2 nM of ³²P labeled DNA probe (TTAGGG)₄ overnight at 42° C in 25 mL of church buffer (7% SDS, 0.25M Na₂PO₄ pH 7.2, 1 mM EDTA, 1% w/v BSA). The membrane was washed with 20 mM Na₂PO₄ pH 7.2, 1% w/v SDS and 1 mM EDTA, exposed to a phosphor imager and the image developed on a typhoon scanner (GE Healthcare). The telomere length was calculated using the software TeloTool (MATLAB) (Gohring et al, 2014 (link))