qPCR Service for Drug Discovery

The real-time quantitative PCR (qPCR; RT-qPCR) service at Reaction Biology provides custom-tailored gene expression analysis and copy number determination including high-quality DNA and RNA isolation. Relative and absolute quantification approaches enable the detection of quantitative differences in mRNA expression of targets, PD markers, or predictive biomarkers. Our qPCR platform is set up to support drug discovery projects performed with cellular and animal models at Reaction Biology, as well as via the investigation of customer samples sent to our lab. 

Our qPCR services support numerous applications in the drug discovery process, such as:

  • Testing for target gene expression after knock-down studies or for identification of suitable tumor cell lines for drug efficacy testing

  • Screening biomarkers to predict a drug’s response in cellular or animal models

  • Determining pharmacodynamic parameters of drugs that affect the expression of specific genes for the determination of time- or dose-dependency and mode of action analysis 

  • Genotyping for verification of stratification markers

Reaction Biology offers complete in-house services comprising sample generation from our cellular and animal models through isolation of DNA and RNA.

Our scientists are available for consultation to meet your research needs with custom-designed project layouts. Our QuantStudio 6 Pro Real-Time PCR System from Applied Biosystems allows absolute and relative quantification assays, multiplex assays, and high-throughput projects. Please reach out to discuss your research goals with our scientists.

Quality data with a quality qPCR setup

QPCRs are performed with the state-of-the-art QuantStudio 6 Pro Real-Time PCR System (Applied Biosystems) for reliable and reproducible gene expression studies.  

All qPCR services are carried out in our qPCR lab with dedicated work areas for DNA and RNA extraction and handling.

Additional qPCR Service Information

qPCR Workflow

qPCR workflow

Sample options

Sample preparation protocols are established for cells and tissues, including tumor material.

Further sample options: 3D cell culture, whole blood, PBMCs, fixed-formalin paraffin-embedded (FFPE) samples, and cell-free samples such as cell culture supernatants, plasma to analyze cell-free, or circulating DNA.

Quality Management

The sample preparation and qPCR analysis will be performed in our dedicated qPCR laboratory, with state-of-the-art equipment and specific work areas for each step.

Furthermore, we use verified protocols and standard procedures to achieve a high standard and minimize the risks of cross-contamination. During all steps, we only use high-quality kits for reliable and reproducible data generation. 

An integrated QC algorithm of the QuantStudio 6 software guarantees optimal performance of the Real-Time PCR reaction.

QC of RNA and DNA samples is performed spectrophotometrically with the UV5 Nano spectrophotometer from Mettler Toledo.

Addition of ROX reference dye and uracil-DNA glycosylase (UDG) to the PCR master mix enable the normalization of the intensity of the reporter fluorochrome and prevent false positives arising from carryover contaminations.

Example: Target gene expression analysis

qpcr target expression

Relative quantification of target gene expression. Gene expression levels of three potential targets have been analyzed to validate a syngeneic mouse model for a subsequent in vivo study.

The comparative Ct method (ΔΔCt) was used for the analysis of relative gene expression. Target gene expression was normalized to the reference gene. The target expression level in healthy lung tissue was used as the calibrator to calculate relative quantitation values (RQ).

The gene expression plot displays the fold change in target expression levels in MC38-CEA cells and MC38-CEA tumor samples relative to C57/Bl6 lung tissue.

Example: Absolute DNA quantification (e.g. as part of biodistribution studies)

qpcr absolute quantification cDMA

Absolute quantification of target DNA. We determine the absolute target mRNA quantity in test samples using the standard curve method. The amplification plot displays the amplification of a 5-fold dilution series of a standard sample (recombinant plasmid).

The x-axis of the standard curve plot represents the logarithm of the diluted DNA quantity, and the y-axis represents the measured quantification cycle (Cq) value.

Example: Analysis of downstream signaling effects after target inhibition

BCL-xL


Investigation of the expression of CDK8/CycC-dependent genes after treatment with two CDK8 inhibitors in HCT116 colon cancer cells.

HCT116 cells were starved overnight, pretreated for 1 hr with different concentrations of compounds or 0.1% DMSO, and stimulated for 2 hrs with 10% FCS.

Analysis of gene expression levels of the CDK8/CycC-regulated Bcl-XL was performed by RT-qPCR. Data were analyzed by the ΔΔCt method using GAPDH expression for normalization. Each column represents the mean ±SE of two biological replicates.

Whereas treatment with compound MC116.4 results in significant downregulation of Bcl-XL mRNA, opposite effects were observed with compound CCT251545.