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. 2019 Nov 27;12:252. doi: 10.3389/fnmol.2019.00252

FIGURE 1.

FIGURE 1

General workflow illustrating procedure for combined UV-LMD and RT-qPCR based mRNA and genomic DNA analysis for individual mouse SN DA neurons. (A) Coronal cryo-sections from juvenile (PN13) mice were stained with cresylviolet (CV) and ethanol-fixed. SN of adult mice (PN90) was in vivo retrogradely traced and coronal cryo-sections were not CV-stained but only ethanol-fixed. SN DA neurons were isolated via UV-LMD into a sterile reaction tube. Subsequently, either genomic DNA was isolated from each sample for qPCR-based quantification of genomic ND1, or a combined lysis and cDNA synthesis protocol was applied, followed by cDNA precipitation for qPCR-based mRNA quantification. A fraction of each cDNA-pool was used for qualitative multiplex-nested marker gene PCR. Note that only samples expressing the correct marker gene profile (TH positive, GAD, CB, GFAP negative) were further analyzed via TaqManTM qPCR, as indicated. GAD, L-glutamate decarboxylase; CB, calbindind28k; GFAP, glial fibrillary acidic protein; TH, tyrosine hydroxylase. Left photographs: Upper: Overview of a juvenile CV-stained wildtype mouse coronal section after UV-LMD of 10 SN DA neurons. The Substantia nigra (SN) is highlighted. Scale bar: 500 μm. Lower: An exemplary juvenile SN DA neuron before (left) and after (right) UV-LMD. Scale bars: 30 μm. Right photographs: Upper: View of an adult in vivo traced injection site (i.e., dorsal striatum), next to an illustration of the respective brain section according to the mouse brain atlas (Paxinos and Franklin, 2013). MC, motor cortex; SSC, somatosensory cortex; CPu, Caudate putamen; NAc, Nucleus Accumbens. Lower: Traced SN DA neuron in fluorescence (left) and brightfield mode (right). Scale bars: 10 μm. (B) Upper: Gel image after capillary electrophoresis of RT-PCR products indicates that all presumed mRNAs are expressed in standard cDNA, derived from PN13 C57BL/6J mouse midbrain tissue in a 1:104 dilution (positive control). Lower: Gel image after capillary electrophoresis of RT-PCR products of an individual SN DA neuron from a WT mouse indicates that all genes analyzed in this study, except for UCP3 and mNCX, are regularly expressed at the mRNA level in individual TH positive SN neurons from WT mice. Note, that we detected positive signals for mNCX only in ∼22% (2 of 9) and signals for UCP2 only in ∼50% (8 of 15) of analyzed WT SN DA neurons.