Molecular Biology

The main focus of molecular biology is the biological activity of cellular molecules such as nucleic acids and proteins that carry out essential biological cell processes. Food constituents and contaminants may have a modulatory effect on biomolecules which are substantial for the cell function and maintenance.

The impact of bioactive food constituents such as anthocyanins or food contaminants like mycotoxins can be measured with different bioanalytical methods. To study the influence of these compounds on mRNA/ miRNA expression or intracellular protein activity the following methods are routinely used in our laboratories:

  • Quantitative real-time PCR: mRNA and miRNA expression levels
  • Bead-based flow cytometry: cytokine secretion levels
  • NF-kappaB reporter gene assay: sequence-specific DNA-binding factor activity

To determine the possible impact of bioactive compounds and contaminants on oxidative stress related target genes, e.g. NQO-1, HO-1 or γ-GCL, we use quantitative real-time PCR (qPCR) or droplet digital PCR (ddPCR). Furthermore we investigate the potential modulatory effect on miRNA expression to elucidate post-transcriptional regulation.

Bead-based flow cytometry is mainly used to examine the effect of bioactive compounds such as polyphenols on inflammatory cytokine secretion. By using a capture sandwich immunoassay the outcome on cells involved in the immune response can be analysed. Additionally, various expression profiles of mRNA or miRNA can be assessed in a single sample (multiplexing).

NF-kappaB is a transcription factor that controls the transcription of cytokine related genes such as IL-6, IL-8 or TNF-α. The NF-kappaB reporter gene assay with luciferase transfected cells is applied to monitor the NF-kappaB signal transduction pathway. Transfected cells are exposed to a stimulus that induces the signaling pathway which triggers the secretion of luciferase. This enzyme in turn catalyzes the oxidation of a specific coelenterazine-based reagent emitting visible blue light with a wavelength between 465-493 nm that can be measured by our plate reader (CytoGen5).