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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.

t 42 C. For the subsequent quantitative RT-PCR analyses 1-2 l of each cDNA-solution were used as templates. The PCR amplification of the cDNA was performed in a LightCycler instrument (Roche) in 20 l reactions using protocols and components of the QuantiTect SYBR Green PCR-Kit (Qiagen). cDNA-specific primers for murine PBGD (porphobilinogen deaminase) were used as model system producing a PCR-fragment of 154 bp. Resulting crossing-point values were used to compare extraction efficiencies of different kits or influences of different staining procedures.
RNA 6000 Pico Assay Protocol
All chips were prepared according to the instructions provided with the RNA 6000 Pico LabChip Kit. In brief, 550 l of the RNA 6000 Pico gel matrix were placed on a spin filter, centrifuged at 1500 g and divided in 65 l aliquots. After addition of 1l RNA 6000 Pico dye concentrate to one gel aliquot, the mixture was vortexed and centrifuged at 13000 g for 10 min. A RNA 6000 Pico chip was filled with gel-dye mix using the chip priming station, followed by addition of Conditioning Solution and Marker. 1 l of RNA 6000 ladder (Ambion) and RNA samples were added in the designated wells, the chip was vortexed for 1 minute and run on an Agilent 2100 bioanalyzer.
Results and Discussion
RNA quality and yield obtained by different isolation kits
All 3 kits tested in this study appeared to work well together with the RNA 6000 Pico assay (figure 2). For the comparison of the different kits we used a standard HE tissue staining and dissected areas corresponding to 1000, 2000 and 3000 cells. Quality and yield of the isolated RNA showed a high variability with respect to the different kits and also within the replicates of one specific method. This may be caused either by differenc
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