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

panel was obtained from Clontech. Based on the LightCycler mRNA Quantification Kit concept, we have developed three new reagent kits for TP, DPD, and TS that are specially designed for usage with fresh-frozen tissues or PET in research applications. The kits are based on a two-step protocol for the quantification of target mRNA expression rates relative to a reference gene (glucose-6-phosphate dehydrogenase, G6PDH) using the LightCycler Instrument and the Relative Quantification Software.
In the first step (reverse transcription), gene-specific primers are utilized to obtain TP, DPD, TS, and G6PDH cDNA in separate reactions. Gene-specific priming is necessary as RNA from PET is degraded to fragments smaller than approximately 200 bp.
The cDNA is then used as a template for quantitative PCR using the LightCycler Hybridization Probe format. Additionally, calibrator RNA from an immortalized cell line is included in the kit and is used as an external standard to allow for relative quantification in each run.

Results and Applications

To validate the new kit concept we have performed three comparative studies:

Proof of principle specific priming

With the standard primer concentration (1 M), the amplification curves in cDNA synthesis tend to decline with decreasing template concentration (Figure 2). Reducing the concentration of the specific primers to 1/5 (0.2 M) leads to reproducible and stable curves in the reverse transcription- polymerase chain reaction (RT-PCR) dilution series, especially with low RNA concentrations (Figure 2). Template RNA in the range of 500 ng/l to as little as 32 pg/l was used. TP data are shown as being representative for DPD, TS, and G6PDH using calibrator RNA as a template. '"/>

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