Sample type, sample preparation and sample QC
Various sample types can be processed with different PCR applications. These include clinical human and preclinical mouse and rat samples, as well as other species on request, ranging from fresh frozen tissues and cell cultures to formalin-fixed, paraffin embedded tissues (FFPE) and different biofluids (e.g. serum, plasma, urine, etc.).
RNA quality and assay specificity is monitored by using microfluidic / capillary gel electrophoresis (Agilent 2100 Bioanalyzer and AATI Fragment Analyzer). Quantification is performed with Qubit Fluorometric Quantitation (ThermoFisher) and by Nanodrop UV-VIS spectrophotometry (ThermoScientific).
Quantitative and digital PCR measurements are carried out using CFX384 touch real-time PCR detection system and QX200 droplet digital PCR system instruments (Bio-Rad).
Quantitative PCR (qPCR) and reverse transcription qPCR (RT-qPCR) are utilized to monitor the amplification of a targeted DNA molecule and for quantification of mRNA, miRNA and lncRNA expression, respectively. DNA binding dye and probe-based detection chemistries are supported.
For PCR assay design, our proprietary primerXL design engine is used, ensuring the selection of the most specific and efficient assays. Data analysis is carried out with our qbase+ software, including an optional geNorm assessment to identify optimal reference genes for normalization, to guarantee accurate results.
Droplet digital PCR
Droplet digital PCR (ddPCR) enables nucleic acid quantification with superior accuracy and sensitivity. This technology excels in gene expression analysis of low abundant genes or small differences, absolute quantification, gene copy number variation, and rare event detection, such as cancer gene mutations.
As absolute concentrations are determined, data can be easily interpreted and compared across different samples and experiments, especially in a clinical setting.
Assays are designed using our proprietary primerXL design engine, ensuring the selection of the most specific and efficient assays.
qbase+ is our commercially available desktop software for qPCR data-analysis on Mac and Windows computers. The user-friendly software is based on peer-reviewed quantification models for PCR efficiency correction, error propagation, inter-run calibration and statistics (Hellemans et al., Genome Biology, 2007). Advanced normalization methods and an improved geNorm algorithm for selection of stably expressed reference genes are built into the software (Vandesompele et al., Genome Biology, 2002).
PCR assay design
Best-in-class PCR assays are designed using our proprietary and ISO17025 accredited primerXL design engine, taking into account secondary structures that impact PCR efficiency, SNPs in primer annealing regions that impact robustness and accuracy, transcript diversity, and ultimate specificity by avoiding putative off-target amplification.