Each animal produced multiple tubes with 10-12 films in each tube. The tubes containing the lysis buffer and films were vortexed to dislodge the captured material from the films. Each tube was adjusted to 350 μl of lysis buffer and then 350 μl of 70% ethanol was added and mixed well. The samples were extracted with the RNAeasy microRNA kit from Qiagen according to the directions. The final eluates of an individual animal were pooled and evaporated in a Speedvac, yielding one preparation per animal. The RNA was suspended in 12 μl of TE (0.01MTris+0.005M EDTA). The quantity of RNA in the resuspended sample was determined with the Ribogreen Quantitation Kit (Molecular Probes, Eugene, OR) or with a Nanodrop Spectrophotometer (ND 1000 V3.3, Wilmington, DE). The integrity of the RNA was examined with the Agilent Bioanalyzer using the pico-chip according to the directions of the manufacturer. This laser capture preparation has been used for examination of several pathways (18 -21 (link)) and it was previously shown to be enriched approximately 7-fold for serotonin neuron-related mRNA (18 ).
The individual animal preparations were split. An aliquot was used for hybridization to the microarray and another aliquot was set aside for qRT-PCR. Two animals per group were used for hybridization and 3 animals per group were used to confirm gene changes with qRT-PCR.
The individual animal preparations were split. An aliquot was used for hybridization to the microarray and another aliquot was set aside for qRT-PCR. Two animals per group were used for hybridization and 3 animals per group were used to confirm gene changes with qRT-PCR.