Additional qPCR Service Information
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 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.
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.
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.