Sodium dodecyl sulfate (sds)

RNA was prepared by lysing 10 million cells in 2 mL of proteinase K solution (500 μg/mL proteinase K [QIAGEN]; 200 mM NaCl [Fisher Scientific]; 200 mM Tris-HCL, pH 7.5 [Wisent]; 1.5 mM MgCl2 [Fisher Scientific]; and 2% SDS [Roche]) for 30 min at 55°C. mRNA was then isolated with 60 mg of oligo(dT) (Sigma-Aldrich) using microcentrifuge spin columns (Ambion). Concentrated mRNA was quantified by absorbance at 260 nm on a NanoDrop 2000c (Thermo Scientific), and quality was verified on an agarose gel by controlling residual 28S/18S contamination. 3 μg of mRNA were separated on a 1% agarose gel containing MOPS (Laboratoire MAT) and 0.62 M formaldehyde (Sigma-Aldrich) and transferred to Immobilon-NY+ nylon membrane (Millipore) by downward alkaline blotting and hybridized with human ³²P-labeled DMPK or GAPDH oligonucleotide probes. Probes were generated by random priming (New England Biolabs) of 100 ng of DMPK cDNA fragments (BglII-SacI) or GAPDH cDNA PCR-amplified fragments (Table S3). Overnight hybridization was conducted at 65°C in hybridization buffer (1X SSPE [Sigma]; 2X Denhardt’s [Sigma]; 10% dextran sulfate [Fisher Scientific]; 1% SDS [Roche]; 100 μg/mL salmon sperm DNA [Ambion]; and probe 1 Mcpm/mL). Relative quantification of mRNA levels on scanned autoradiograms (600 dpi) was determined by densitometry using ImageJ 1.47 software.

Total RNA was purified from mouse tibialis anterior, soleus, triceps, quadriceps, latissimus dorsi, diaphragm, heart, and brain after flash freezing in liquid nitrogen. The isolation process started with a crude homogenization of 25 mg of tissue in proteinase K solution (10 mM Tris-Cl, pH 7.5 [Wisent]; 10 mM EDTA [Sigma-Aldrich]; 2% SDS [Roche]; 500 mM NaCl [Fisher Scientific]; 1.5 mM MgCl2 [Fisher Scientific]; and 500 μg/mL proteinase K [QIAGEN]), followed by complete homogenization with a TissueLyser LT (QIAGEN) using a ball bearing. After a 30-min incubation at 55°C, standard QIAzol (QIAGEN) protocol for tissue extraction was performed along with RNA quantification with a NanoDrop 2000c (Thermo Scientific), and RNA integrity was verified on a 2100 Bioanalyzer (Agilent Technologies). cDNA was then produced using Quantitect Reverse Transcription (QIAGEN) on 200 ng of RNA, and RT-qPCR was used to determine mRNA levels for DMPK and Dmpk mRNA, with Hprt1, Rpl13a, and Tbp RNAs as normalization controls (primers in Table S3) on a LightCycler 480 II (Roche) using SYBR Green I Master hot start reaction mix.