( ...)Gene-Expression Analysis of TP, DPD, and TS Using,,,the LightCycler mRNA Quantification KitsPLUS

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The new primer concentration also extends the dynamic measuring range of the target and reference and improves sensitivity compared to random hexamer priming (Figure 3 a-c). Table 3d numerically shows the crossing point (Cp) ranges for all four genes TP, DPD, TS, and G6PDH.



The human RNA panel (healthy tissue)

We have also analyzed both priming concepts using a panel of 21 human RNA samples from healthy tissues. We observed a slight shift to smaller Cp values with specific priming for all four parameters TP, DPD, TS and G6PDH indicating the higher sensitivity of the new concept (Figure 4). These shifts depend on the parameter and the analyzed tissue type. Also, the comparison of the relative ratios reveals a tendency for slightly higher ratios with specific priming. This is caused by different Cp shifts between calibrator and sample RNA. Nevertheless, the expression rates are comparable and within a common range.



Comparison of fresh-frozen tissue vs. PET

For this study, we analyzed xenografts from human immortalized colon tumor cell lines HT29 and SW62 with the new kit concept. Total RNA was isolated from PET in 4 x 5-m slides and from matched fresh-frozen tissues in two 20-mg slices according to the High Pure RNA Paraffin Kit protocol. As depicted in Figure 5, the relative expression rates of TP, DPD, and TS are comparable in both tissue types.



The new kit concept with specific priming is superior to former hexamer protocols and shows comparable Cp values and ratios. The linear measuring ranges were extended and the sensitivity of analysis was improved for all three target genes, a
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