Quantitative PCR (qPCR)
qPCR is a technique that allows scientists to monitor the amplification of a targeted DNA throughout the PCR and not at the end as with traditional PCR.
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.
Application of qPCR:
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.