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Analytical Validation

Analytical Validation

Analytical expertise for DNA, RNA & proteins.

Analytical Validation

In the Analytical Validation Department, we address technical research questions in the field of nucleic acids and proteins. With our experienced team specialized in numerous PCR technologies, primer design, protein analyses and purification strategies, we provide reliable solutions for internal and external cooperation partners to improve TRON’s excellent scientific approaches.

The Analytical Validation Department combines information on DNA and RNA level with protein analysis data and thus opens the way for a wide range of investigative and confirmative methods and assays addressing questions related to both, molecular biology and biochemistry, and improves our excellent scientific approaches.

  • Dr. Stefanie Hubich-Rau

    Director of Analytical Validation

Our vision

We transform scientific expertise, cutting-edge technologies, and molecular data into groundbreaking insights that sustainably accelerate scientific progress and shape the future of research.

Medical Biochemistry Unit

  • Dr. Stefanie Hubich-Rau

    Head of Functional Unit Medical Biochemistry

  • René Becker

    Deputy Head of Medical Biochemistry

Our activities and competences

Our methods and platforms

Protein separation

We perform classical polyacrylamide and capillary electrophoresis (SDS-PAGE & CE) followed by Western blotting (manual or automated by Jess).

Protein detection

Protein detection by Western blot using chemiluminescence and fluorescence imaging systems. Reversible total protein staining serves as a loading control.

Antibody bank

We offer a broad portfolio of commercial antibodies that have been validated using overexpressed cell lysates and/or siRNA-based knockdown experiments.

Cell lysate bank for WB

More than 300 quality-controlled cell lysates derived from standardized cell lines are available and can be used as controls across a broad range of assays and analytical workflows.

Recombinant protein expression

Production of recombinant proteins in bacterial (E. coli) and insect (Sf9) cell expression systems.

Protein purification

Proteins are purified on an ÄKTA go™ chromatography system using affinity, size-exclusion, and ion-exchange chromatography techniques to achieve high purity and quality.

Publications

The co-delivery of microRNAs (miRNAs) and protein-coding RNA presents an opportunity for a combined approach to gene expression and gene regulation for therapeutic applications. Protein delivery is established using long mRNA, self-, and trans-amplifying RNA (taRNA), whereas miRNA delivery typically uses short synthetic oligonucleotides rather than incorporating it as a precursor into long RNA. Although miRNA delivery into the cell cytoplasm using long genomes of RNA viruses has been described, concerns have remained regarding low processing efficiency. However, miRNA precursors can be released from long cytoplasmic alphaviral RNA by a cytoplasmic fraction of Drosha. taRNA, a promising vector platform for infectious disease vaccination, uses a nonreplicating mRNA expressing an alphaviral replicase to amplify a protein-coding short transreplicon-RNA (STR) in trans. To investigate the possibility of simultaneously delivering protein expression and gene silencing, we tested whether a taRNA system can carry and release functional miRNA to target cells. Here, we show that mature miRNA is released from STRs and silences specific targets in a replication-dependent manner for several days without compromising the expression of STR-encoded proteins. Our findings suggest that incorporating miRNAs into the taRNA vector platform has the potential for gene regulation alongside the expression of therapeutic genes.

PLAC1 (placenta enriched 1) is a mammalian trophoblast-specific protein. Aberrant expression of PLAC1 is observed in various human cancers, where it is involved in the motility, migration, and invasion of tumor cells, which are associated with the phosphoinositide 3-kinase (PI3K)/AKT pathway. We previously demonstrated that AKT activation mediates the downstream effects of PLAC1; however, the molecular mechanisms of PLAC1-induced AKT-mediated tumor-related processes are unclear. We studied human choriocarcinoma and breast cancer cell lines to explore the localization and receptor-ligand interactions, as well as the downstream effects of PLAC1. We show secretion and adherence of PLAC1 to the extracellular matrix, where it forms a trimeric complex with fibroblast growth factor 7 (FGF7) and its receptor, FGF receptor 2 IIIb (FGFR2IIIb). We further show that PLAC1 signaling via FGFR2IIIb activates AKT phosphorylation in cancer cell lines. As the FGF pathway is of major interest in anticancer therapeutic strategies, these data further promote PLAC1 as a promising anticancer drug target.

PCR Analytics Unit

  • Martin Suchan

    Head of Functional Unit PCR Analytics

Our activities and competences

Our methods and platforms

PCR technologies

Different Real-Time PCR formats are used for expression analysis, transcript differentiation, SNP analysis, CNV, genotyping, fusion and splice variants analysis. Our instruments’ capacities range from 96-, to 384-Well and microfluidic formats with thousands of parallel reactions.

Analysis of PCR products

Classic end-point PCR and gel electrophoresis are done for recovaery of distinct bands and subsequent confirmation of the PCR product by sequencing, allowing analysis of sequence variation, e.g. insertions, deletions, substitutions, methylation.

Droplet digital PCR technology

Droplet digital PCR (ddPCR) is applied for high-sensitive, absolute quantification of single DNA copies, used for expression, SNP, genotyping, CNV and rare event analysis, for example.

cDNA & RNA bank

Large selection of cDNAs and RNAs that are immediately available for PCR application.

Primer design & database

Design and validation of PCR primers for human and mouse transcripts as well as tracking of verified expression assays. Custom assay design for various origins, e.g. virus, endogenous constructs. Database of primers for genomic analyses or construct detection.

Microfluidic qRT-PCR (Standard BioTools) database

Extensive Ct-value, QC-validated database enabling quick querying of expression values from in-house high throughput microfluidic qRT-PCR experiments.

Publications

T cells directed against mutant neo-epitopes drive cancer immunity. However, spontaneous immune recognition of mutations is inefficient. We recently introduced the concept of individualized mutanome vaccines and implemented an RNA-based poly-neo-epitope approach to mobilize immunity against a spectrum of cancer mutations. Here we report the first-in-human application of this concept in melanoma. We set up a process comprising comprehensive identification of individual mutations, computational prediction of neo-epitopes, and design and manufacturing of a vaccine unique for each patient. All patients developed T cell responses against multiple vaccine neo-epitopes at up to high single-digit percentages. Vaccine-induced T cell infiltration and neo-epitope-specific killing of autologous tumour cells were shown in post-vaccination resected metastases from two patients. The cumulative rate of metastatic events was highly significantly reduced after the start of vaccination, resulting in a sustained progression-free survival. Two of the five patients with metastatic disease experienced vaccine-related objective responses. One of these patients had a late relapse owing to outgrowth of β2-microglobulin-deficient melanoma cells as an acquired resistance mechanism. A third patient developed a complete response to vaccination in combination with PD-1 blockade therapy. Our study demonstrates that individual mutations can be exploited, thereby opening a path to personalized immunotherapy for patients with cancer.

Cancer-associatedgene fusions are a potential source for highly immunogenic neoantigens, but thelack of computational tools for accurate, sensitive identification of personalgene fusions has limited their targeting in personalized cancer immunotherapy. Herewe present EasyFuse, a machine learning computational pipeline for detectingcancer-specific gene fusions in transcriptome data obtained from human cancersamples. EasyFuse predicts personal gene fusions with high precision andsensitivity, outperforming previously described tools. By testingimmunogenicity with autologous blood lymphocytes from patients with cancer, wedetected pre-established CD4+ and CD8+ T cell responses for 10 of 21 (48%) and for 1 of30 (3%) identified gene fusions, respectively. The high frequency of T cellresponses detected in patients with cancer supports the relevance of individualgene fusions as neoantigens that might be targeted in personalizedimmunotherapies, especially for tumors with low mutation burden.

Major histocompatibility complex (MHC) class I antigen presentation deficiency is a common cancer immune escape mechanism, but the mechanistic implications and potential strategies to address this challenge remain poorly understood. Studying b2-microglobulin (B2M) deficient mouse tumor models, we find that MHC class I loss leads to a substantial immune desertification of the tumor microenvironment (TME) and broad resistance to immune-, chemo-, and radiotherapy. We show that treatment with long-lasting mRNA encoded interleukin-2 (IL-2) restores an immune cell infiltrated, IFNg-promoted, highly proinflammatory TME signature, and when combined with a tumor-targeting monoclonal antibody (mAB), can overcome therapeutic resistance. Unexpectedly, the effectiveness of this treatment is driven by IFNg-releasing CD8+ T cells that recognize neoantigens cross-presented by TME-resident activated macrophages. These macrophages acquire augmented antigen presentation proficiency and other M1-phenotype-associated features under IL-2 treatment. Our findings highlight the importance of restoring neoantigen-specific immune responses in the treatment of cancers with MHC class I deficiencies.

The co-delivery of microRNAs (miRNAs) and protein-coding RNA presents an opportunity for a combined approach to gene expression and gene regulation for therapeutic applications. Protein delivery is established using long mRNA, self-, and trans-amplifying RNA (taRNA), whereas miRNA delivery typically uses short synthetic oligonucleotides rather than incorporating it as a precursor into long RNA. Although miRNA delivery into the cell cytoplasm using long genomes of RNA viruses has been described, concerns have remained regarding low processing efficiency. However, miRNA precursors can be released from long cytoplasmic alphaviral RNA by a cytoplasmic fraction of Drosha. taRNA, a promising vector platform for infectious disease vaccination, uses a nonreplicating mRNA expressing an alphaviral replicase to amplify a protein-coding short transreplicon-RNA (STR) in trans. To investigate the possibility of simultaneously delivering protein expression and gene silencing, we tested whether a taRNA system can carry and release functional miRNA to target cells. Here, we show that mature miRNA is released from STRs and silences specific targets in a replication-dependent manner for several days without compromising the expression of STR-encoded proteins. Our findings suggest that incorporating miRNAs into the taRNA vector platform has the potential for gene regulation alongside the expression of therapeutic genes.