( Laser capture of as many neurons labele...)Laser Capture Microdissection (LCM) and Expression Profiling of Long-Range Projection Neurons

Laser capture of as many neurons labeled with a single color as possible was performed within a time period of 45 minutes from when each slide was thawed. CapSure HS LCM caps were used with Pixcell IIe settings as follows: 450-700 s duration, 60-85 mW power, 15 m laser diameter. These settings permitted the capture of single cells with a diameter ranging from 7-12 m, close to the diameter of HVC projection neuron cell bodies. Microdissection of 1500 neurons projecting to area X and 2000 neurons projecting to nucleus RA from within the boundaries of HVC was performed from each cell type for each animal. This yields approximately equal amounts of amplified RNA for the two cell types.

4. When all desired cells of one color were captured on an HS LCM cap, 15 L of extraction buffer from the PicoPure RNA isolation kit was added to each cap. Each cap was then incubated at 42C as per the kit instructions. The tubes containing the extracts were spun down and stored -80C until ready for purification (no more than 1 week).

C. RNA purification and amplification
1. Samples from a single projection neuron type, from a single animal, were pooled by combining all 15 L extracts. An equivalent volume of 70% EtOH was added to the pooled sample, and RNA isolation was continued according to the instructions in the PicoPure kit, including the on-column DNase digestion detailed in the user guide Appendix A. Each RNA sample was eluted in 11 L elution buffer.

2. RNA was amplified through two rounds with the RiboAmp HS kit according to the manufacturers instructions.

3. aRNA quality was evaluated on the Agilent Bioanalyzer 2100 using the Nanochip, and the aRNA yield was measured on the NanoDrop ND-1000 spectrophotometer. Average yield from the captured songbird bird material was approximately 70-90 g after two rounds of amplification with the RiboAmp HS kit.

4. aRNA was stored at -80C until subs
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