P a l m laser microdissection system

RNA from myocardial sections was captured by LCM using a Zeiss P.A.L.M. laser microdissection system as previously described by authors herein (Thorp 2011 (link)). Total RNA was isolated using the RNAqueous-Micro kit from Ambion and reverse-transcribed into cDNA using Superscript III First-Strand Synthesis Mix (Invitrogen). For semi-quantitative and Quantitative RT-PCR: Hearts were snap-frozen for RNA. Total RNA was extracted using the RNeasy kit (Qiagen). cDNA was synthesized from 4 μg of total RNA using oligo (dT) and Superscript II (Invitrogen). cDNA was subjected to quantitative RT-PCR amplification using a SYBR Green PCR Master Mix (Applied Biosystems).

35 μm sections of labeled brains were stained for EGFP, and mounted on membrane PEN slides (Carl Zeiss). Labeled neurons were visualized, and captured into adhesive cap tubes (Carl Zeiss) using a PALM laser microdissection system (Carl Zeiss) using a uv laser to cut and flip tissue samples directly into inverted adhesive caps placed above slides. RNA was extracted using an RNeasy FFPE kit (QIAGEN). Reverse transcription was performed using random hexamers and Thermoscript reverse transcriptase (Fisher Scientific). Real time PCR was performed using SYBR® Green PCR Master Mix (Thermo Fisher) on a Realplex⁴ cycler (Eppendorf). We confirmed the specificity and size of the amplicons by running the PCR product on agarose gels, and by melting curve analysis. For Klhl14-overexpression experiments, data were analyzed using an unpaired two-sided t test. The PCR primers are listed in Key Resources Table.

RNA from myocardial sections was captured by LCM using a Zeiss P.A.L.M. laser microdissection system as previously described (21 (link)). Total RNA was isolated using the RNAqueous-Micro kit from Ambion (Waltham, Massachusetts) and reverse-transcribed into cDNA using SuperScript III First-Strand Synthesis Mix (Invitrogen, Carlsbad, California).