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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.

). In the case of MB staining, ambiguous results were observed (average yield: approx. 730 pg; partially degraded RNA; figure 3D). The 28 S band is almost entirely missing, which could be due to RNA degradation or due to a salt content that exceeds the salt specifications of the Pico kit. RNA-extraction with the kit of manufacturer B from the same sections with all four staining methods yielded slightly different relations (see in RT-PCR results below), and overall lower concentration levels as expected from the comparison of the kits from above. It should be noted that this short investigation does not put a value on the use of certain cell stains for work with microdissected material but rather points out the value of the RNA 6000 Pico kit for optimization of experimental protocols.

Correlation of the results obtained by the RNA Pico Assay to real-time RT-PCR
Comparison of RNA extraction kits:
All 3 sample extraction kits that were tested yielded good quality RNA that could be used for RT-PCR (figure 4). For the comparison we used HE-stained sections to microdissect and catapult areas of 1000 cells each (3 replicates per sample). After RNA isolation with the three different kits real-time RT-PCR with the LightCycler was performed. The amount of cDNA used as template was half of the RNA used for the 2100 bioanalyzer analysis (fig. 4A). The specificity of the PCR-fragments was proven by the melting curve (figure 4B). The crossing points (Cp)* of the growth curves (figure 4C) being a scale for the initial specific RNA-amount were close together (about one cycle distance each) correlating to the Bioanalyzer values although Manufacturer A was overestimated there probably due to the DNA-contamination (figure.1).


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