Palmrobo software 4

Fetal brains were cut into 16 μm sections on PALM Membrane Slides (415190-9081-000, MembraneSlide, Carl Zeiss, North York, ON, Canada). Cryostat blades and workstations were cleaned with RNaseZap (Applied Biosystems, Life Technologies, Carlsbad, CA, USA) to prevent RNA degradation. 10 sagittal sections were used for LCM. Duration was limited to <30 min per slide to prevent RNA degradation. For each fetal brain section, the hindbrain was localized by morphology under the Axio Observer.Z1 confocal microscope (Zeiss; Thornwood, NY). Conservative regions of interest encompassing approximately 100 cell nuclei were microdissected (energy: 50–53; focus: 73) using the PALM Microbeam system and PALMRobo software 4.3 (Zeiss; Thornwood, NY) and immediately catapulted into an AdhesiveCap 200 microcentrifuge tube (415190-9181-000, Carl Zeiss, North York, ON, Canada Zeiss). RNA isolation was performed immediately as described above, with the RNeasy Micro Plus kit (Qiagen, Gaithersburg, MD, USA). Genomic DNA was removed using gDNA eliminator columns (Qiagen, Gaithersburg, MD, USA). Total RNA was amplified and reverse-transcribed using the QuantiTect Whole Transcriptome Amplification kit (Qiagen, Gaithersburg, MD, USA). 100 ng cDNA was used for qPCR, according to the methods described above.