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Quality assurance of RNA derived from,,,laser microdissected tissue samples,,,obtained by the PALM MicroBeam System,,,using the RNA 6000 Pico LabChip kit.

er Hematoxylin/Eosin (HE), Nuclear Fast Red (NFR), Methylgreen (MG) or Methylene Blue (MB) staining was applied. After staining and a short increasing ethanol series the sections were air-dried and either used immediately or refrozen at 20 C for up to 2 days. The PALM Micro- Beam System was used to precisely excise the selected tissue areas with the UV-A-laser and then to catapult the areas of interest contact free against gravity into collecting caps filled with 12 l of RNase-free water. For lysis the isolated material was then immediately mixed by inversion with the respective lysis buffers for RNA extraction (see below).
RNA Purification
For this study RNA isolation kits from 3 different manufacturers were used. Two are based on a column-purification, while the third one uses an extraction-/precipitation- based approach. RNA cleanup was carried out according to the manufacturers protocols with the only variation that uniformly a volume of 30 l of elution buffer or water was used in the final step to collect the purified RNA. For the column-procedures the elution process was repeated once with the primary eluate of 30 l to improve the yield. The scope of this work was the demonstration of feasibility to perform RNA sample quality control from microdissected material and not the evaluation of commercially available RNA extraction kits. Therefore, the manufacturers are not explicitly named here, but instead referred to as manufacturer A, B, and C.
RT-PCR
Selected RNA samples were reversely transcribed with the First Strand cDNA Synthesis Kit for RT PCR (AMV) cDNA Synthesis Kit for RT-PCR (Roche) according to the manufacturers protocol. Briefly, 5-8 l of each RNA solution were transcribed by AMV-RTase with random primers in a total volume of 20 l for one hour a
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IHC Polymer Detection Kit (Rabbit DAB)150 slides...
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Bovine Serum...
Healthy animals 10-32 weeks of age, mixed gender and breed; collected on wet ice following USDA guidelines, using Alsevers solution at 1:2 dilution...
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