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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.

Comparison of staining procedures
In this test serial tissue sections were stained using either Hematoxylin/Eosin (HE), Nuclear Fast Red (NFR), Methylgreen (MG) or Methylene Blue (MB). Areas corresponding to 2000 cells were dissected, the RNA purified using Manufacturer B (3 replicates each) and analyzed with the 2100 bioanalyzer (figure 5A) and the Light-Cycler as above. The cDNA input for the PCR corresponded to a fourth of the RNA amount used for the 2100 bioanalyzer run. The melting curve proves the specificity of the products (figure 5B), while the growth curve reflects the starting amount of RNA (figure 5C). In contrast to the test with Manufacturer C (see above) NFR was the optimal stain, though the Cp-values lay very close together (only half a cycle difference) except for MB which showed worse results. (CpNFR: 34,7; CpHE: 35,2; CpMG: 35,8; CpMB: 37,6). This real-time RT-PCR results again correlated well with the 2100 bioanalyzer values.

Conclusion
RNA derived from laser-microdissected tissue isolated by the PALMMicroBeam system is of high quality and can be analyzed efficiently using the RNA 6000 Pico assay with the Agilent 2100 bioanalyzer. RNA-purification kits of different manufacturers and various common staining procedures have been tested and were compatible with the Pico Assay. Nevertheless, it seems to be advisable that any extraction protocol and staining method should be tested in combination to find the optimal procedure for tissue microdissection experiments. The RNA 6000 Pico kit is well suited to show differences in RNA quality and, therefore, is an ideal tool to optimize experimental conditions. Its unprecedented sensitivity allows for the first time quality control in the context of microdissection experiments
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