( ards. Standard curves from this materia...)Laser Capture Microdissection (LCM) and Expression Profiling of Long-Range Projection Neurons

Results
We have marked two types of long-range projection neurons in the zebra finch brain with fluorescent tracers, and then captured 1500-2000 of these neurons one-by-one using a Pixcell IIe (Fig. 1 a-e). RNA from these captures was pooled by neuron type, amplified through two rounds using the RiboAmp HS kit, and then assessed for quality on an Agilent Bioanalyzer. High quality amplified RNA profiles were consistently obtained from each of 14 birds (Fig. 2). The aRNA from each projection neuron type was compared, using a withinanimal design, by applying Cy dye-labeled cDNA to cDNA microarrays made from whole zebra finch brain.

More than 98% of the genes were found to be expressed at similar levels in this comparison, as assessed by a cutoff of 2- fold differential expression at least 50% of the time across all birds in the experiment. This arbitrary fold-change cutoff was supplemented with a statistical analysis (Significance Analysis of Microarrays) that also showed more than 90% of the genes in the two neuron types to be expressed at similar levels. We focused on the few genes that showed the most consistently dramatic differences in expression, in an attempt to develop markers of these projection neuron subtypes. Using in situ hybridization (Figure 3), we were able to validate 7 of 8 genes meeting a criterion of 2-fold or more differential expression in at least half of the microarray hybridizations. ISH reveals that not every neuron of a given subtype expresses the gene of interest (Fig. 3 b and d), indicating further heterogeneities within a projection neuron class. This is not surprising, given that we pooled hundreds of each type of neuron in our microdissections. Three of these 8 most differentially expressed
'"/>

Source: