qPCR Service for Drug Discovery

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.

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.

Relative quantification of drug 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 C_t method (ΔΔC_t) 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.

Absolute quantification of drug target mRNA. 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.

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 ΔΔC_t 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.

Digital PCR (dPCR) Service

The digital PCR (dPCR) service from Reaction Biology allows for precise and sensitive quantification of target molecules such as nucleic acids as well as highly sensitive analysis of complex cellular events such as single-nucleotide mutations, providing important insights into tumor genetic composition. The primary goal of dPCR is to provide accurate quantification of target molecules in a sample without the need for standard curves or external references. dPCR works by dividing a sample into thousands of individual reaction chambers, each of which can support PCR amplification.

Following separation, PCR amplification occurs independently in each chamber containing the target sequence of interest. The QIAcuity One 5plex digital PCR  system is used for dPCR analysis, and the final analysis is done with either the TaqMan assay and primers plus SYBR Green. Chambers that detect a signal are scored as positives, and chambers without signal are scored as negative.

dPCR is a highly sensitive PCR method particularly well suited for:

• Viral load quantification
• Gene expression analysis (for low changes in expression)
• Rare mutation detection
• Liquid biopsies

dPCR Assay Setup

PCR sample is partitioned into thousands of individual reaction chambers, each of which can support PCR amplification independently.

The PCR reaction occurs in thousands of chambers, and the results are analyzed by imaging for positive signals.

Copy number per µl is determined without the need of traditional statistical methods as plotting a standard curve.

Advantages:

• Absolute quantification
• High precision and sensitivity
• Tolerance to PCR inhibitors
• High reproducibility

Sample Data

Mice were intravenously infected with different dosages of tissue-specific virus (viral genomes [vg]). Tissue of interest was isolated, DNA was purified and analyzed for viral copies by QIAcuity One 5plex digital PCR system (Qiagen) using viral promoter-specific TaqMan probes.