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Quantitative PCR

Aurelia Bioscience is now able to offer quantitative PCR (qPCR) service to our clients. Using our Applied Biosystems™ QuantStudio™ 6 Flex Real-Time PCR system, we are able to investigate quantitative differences in mRNA expression in relation to pathways and targets identified as being of interest to clients. The QuantStudio™ offers a high throughput real-time PCR platform (with a 384 well block) with sophisticated data analysis software. Designed to easily manage large data sets, the embedded gene expression study application can analyse >100 runs, while heat maps and scatter plots provide quick checks on data quality.

Example of Application

Using the QuantStudio™, a client MOA study was designed to determine the activity of compounds inhibiting target gene expression, relative to a ‘house-keeping’ gene. Cells plated in 96 well format were treated with compounds at varying concentrations. The cells were then processed using Cells-to-CT™ kits (Thermofisher), which provide a complete workflow for real-time qRT-PCR analysis directly from cultured cells without RNA purification. Cultured cells are lysed while removing genomic DNA (gDNA) and preserving RNA integrity, the kits contain reverse transcription (RT) reagents for cDNA synthesis and the master mix for rtPCR analysis. Multiplex qPCR reactions (4 replicates/sample) were performed using optimised primers.

Figure 1:Target mRNA vs ‘housekeeping’ mRNA amplification in human cell line samples using 1 step cell to PCR methodology.
Multiplex qPCR reactions (4 replicates/sample) were performed using appropriate primers.

Figure 3: Inhibition of target gene expression in a human cell line.
The samples were analysed using QuantStudio™ 6 Flex software, and the Relative Quantitation RQ values were used to determine IC50 values with GraphPad Prism 7 curve fitting.

Figure 2: Relative Quantification of target gene expression in human cell lines following treatment with different compounds.
The comparative CT method (ΔΔCT) was used for analysis of relative expression. Target gene expression was normalised to the housekeeping internal control gene. Target expression in unstimulated DMSO control sample was used as the calibrator to calculate Relative Quantitation (RQ) values.

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