Molecular Diagnostic Laboratory
Department of Molecular Medicine,
Aarhus University Hospital Skejby
Functional Genomics and Marker Validation
Aims and Vision
Within the last years, researchers at the Department of Molecular Medicine
(MOMA) have identified a number of single molecules dysregulated in colorectal
cancer, bladder cancer or prostate cancer as well as signatures which can
predict the disease course or treatment response.
Molecules were identified by extensive profiling studies on hundreds of tissue samples by transcript microarray profiling using Affymetrix GeneChips U133plus2.0, microRNA expression profiling using Exiqon Locked Nucleic Acid (LNA™) detection technology and/or Applied Biosystems TaqMan® Low Density Array (microfluidic cards), Methylation profiling using Illumina’s HumanMethylation27 DNA Analysis BeadChip or SNP analyses using Affymetrix Genome-Wide Human SNP Array 6.0.
On the one hand, signatures will be validated in large multicenter retrospective and prospective studies with the aim to use theses signatures for clinical application (see organ sites). On the other hand, functional analyses will be performed on selected molecules with clinical relevance. Those molecules comprise e.g. mRNA transcripts encoding proteins as well as microRNAs.
Each project has its origin in the box headed “Biobank”,
where samples are collected and nucleic acids are extracted, quality
controlled and stored (for detailed information, see CRC or bladder
Expression of gene encoded proteins and their localization within the tumor cell is analyzed by immunohistochemistry using cryosections in Tissue Tek or FFPE tissue specimen.
For protein detection, antibody specificity is checked on Western
Blots using cell lines overexpressing the molecule of interest.
In situ hybridization (ISH) using LNA probes is used
to localize microRNAs within the tumor cells.
Cell Line authentication
In case of microRNAs cells are transfected with miR-precursors or Anti-miRs, miRNA Inhibitors targeting human miRNAs (Exiqon). Successful up- or downregulation is monitored by RTPCR (Taqman) and Western blotting for protein.
Cells with an overexpressed or silenced molecule of interest are continuously monitored by Real Time Cell Analysis RTCA (Roche X-celligence) over a period of up to 5 days to identify whether the molecule of interest impacts cellular processes, e.g. increases proliferation or causes cell death. Standard cellular assays monitoring proliferation, viability or cytotoxicity are performed.
Data analyses in MOMA are performed on a Dual Plate RTCA instrument,
offering highest possible flexibility for several different approaches
Fluorescence microscopy visualizes the cellular localization of proteins within living or fixed cells from cell lines, from cryosections or from FFPE tissue samples.
addition to the above mentioned analyses as e.g. RTCA, protein work
and microscopy, a large number of our functional cellular analyses
are performed in 24-96 well microtiterplates using a Biotek Synergy
For Pathway analyses, to gain insight into networks or to identify
downstream target molecules, cell extracts from manipulated cells, overexpressing
a molecule of interest or with a knock down, are subjected to Microarray
expression profiling. Bioinformatic data processing is followed by application
of Ingenuity Pathway analyses software to identify pathways or networks
addressed in common for both, the tumor samples as well as the manipulated
cell lines. (K. Demtröder, unpublished data)
Future Research Activities