Next-Generation Diagnostics

The Next-Generation Diagnostics Unit identifies and characterizes disease-associated nucleic acids for their application as diagnostic markers and therapeutic targets, and develops experimental methods for this purpose. A special focus is placed on the establishment of suitable sequencing protocols, the performance of microarray analyses as well as the development of special enrichment processes to characterize diagnostic markers.

Another field of activity of the unit is the identification and functional characterization of non-protein coding RNAs (ncRNAs), hence that part of the transcriptome of a cell that carries no signal for translation into proteins. The ncRNAs are very specifically regulated, show high cell-type specificity and occur surprisingly often in association with diseases. The identification of clinically-relevant ncRNAs opens completely new perspectives for the development of therapeutic tools for the effective and targeted attack on sickened cells.

Analysis methods for the biological marker validation and test development

The unit has a special focus on the continuous establishment of new and the adaptation of existing analysis methods for particular questions of biomarker validation. The work primarily involves next-generation sequencing, microarrays analyses as well as PCR-based processes. Solutions for the development of marketable test systems are moreover in development, in close collaboration with the RNA Biomarker Unit in order to precisely define the requirements for the respective biological marker groups.

Next-generation sequencing

The Next-Generation Diagnostics Unit uses the HiSeq 2500 System as well as the MiSeq System from Illumina for next-generation sequencing. These sequencing platforms are applied in a broad spectrum of methods. Aside from performing genome- and transcriptome-wide sequencing, the unit’s focus is on establishing and further developing special sequencing protocols, for instance for DNA / RNA capture sequencing, amplicon sequencing, identifying SNPs and mutations as well as for characterizing small genomes. The outstanding connection to the Bioinformatics Unit subsequently permits a highly qualitative evaluation of this sequencing data.

Microarray analyses

A microarray platform from Agilent is available for the highly parallelized performance of gene expression analyses. Apart from the microrarrays for mRNA, Exon, miRNA and ncRNA analyses offered by Agilent, user-defined microarrays for special questions can be designed in collaboration with the Bioinformatics Unit.

Interaction studies

The Next-Generation Diagnostcs Unit has built up an extensive range of methods to obtain insights into the cellular interactome. The SILAC method has been successfully employed for protein-protein interaction studies. Furthermore, chromatin immunoprecipitation (ChIP) and RNA immunoprecipitation (RIP) have been conducted to examine the interplay of proteins with DNA or RNA. Likewise protocols were established to identify RNA-binding partners to RNA, at the DNA and protein level, which currently are especially being used in the search for new ncRNA-binding partners.

RIBOLUTION

The "RIBOLUTION – integrated platform for the identification and validation of innovative RNA-based biomarkers for personalized medicine" project is a research association supported by the “Fraunhofer-Zukunftsstiftung” (Fraunhofer Future Foundation). The project is coordinated by Prof. Dr. Friedemann Horn at Fraunhofer IZI.

New RNA biomarkers for complex disorders (e.g. oncological, chronic-inflammatory and degenerative disorders) are being identified in the RIBOLUTION project by genome-wide screening processes. RIBOLUTION also includes so-called non-coding RNAs whose significance as potential biological markers have only been investigated in the recent past. Additionally, the process of the biological marker screening by RIBOLUTION is optimized and perfected with the help of technical innovations.

Subsequent to the screening phase, potential biomarkers are evaluated with regard to their diagnostic and prognostic significance. Biomarkers with the ability to indicate a disorder or its course or forecast the response to therapies are of special interest.

Endocarditis

In cooperation with the Heart Center Leipzig and the Institute for Microbiology of the University of Leipzig, the topic of improved pathogen diagnostics on infectious endocarditis is being investigated.

By means of genome-wide sequencing, pathogens are identified both directly on the infected cardiac valves as well as in the blood of the patient and compared with the findings of the microbiological analysis laboratory.

In addition to the already available microbiological analyses, the objective is to devise an optimized treatment strategy for every individual patient.

Mammary carcinoma

In a joint project with the University Gynecological Hospital Essen, genes are examined that are relevant for the formation and metastatic spread of mammary carcinoma, with respect to their expression pattern and the occurrence of mutations.

The unit developed a special DNA capture approach that permits disease-relevant DNA sections from primary tumors and metastases of patient to be selectively enriched and subsequently sequenced. Simultaneously at the Essen Hospital, circulating tumor cells (CTCs) are isolated from patient blood and the expression of mammary carcinoma-relevant genes examined by means of PCR.

The data from these studies can show how primary tumors and metastases differ in their mutation and expression patterns. Moreover, the project investigates whether CTCs can be employed for the diagnosis of mammary carcinoma and deliver indications for a target-specific treatment. As a result, complicated surgical removal of tumor tissues could become redundant.

Functional analyses for the role of non-coded RNAs in multiple myeloma, glioblastoma and Alzheimer's disease

In the past years, genome-wide next-generation sequencing has identified a multitude of non-protein coded RNAs (ncRNAs). Many of these ncRNAs apparently influence the formation and course of diseases. Nevertheless, a large part of these transcripts are hardly functionally characterized.

The main focus of this project therefore is comprehensive functional analyses of these ncRNAs in various immunological and neurologically relevant cell systems. With the selective knockdown of an ncRNA, subsequent examinations allow the determination of their influence on vitality, apoptosis rate, proliferation, adhesion and cell migration. Furthermore, the expression and localization of ncRNAs within tissues, but also on the single-cell level, can be visualized and quantified. In vivo interaction studies have deepened the identification of ncRNA-binding partners on the RNA, DNA and protein level. The objective is to clarify the molecular and cellular therapeutic mechanism of ncRNAs to better understand their role in diverse disease models and to establish possible therapeutic approaches.

Completed project: Diagnostics of circulating tumor cells

This SAB- funded R&D association project was carried out by ApoCell Europe GmbH together with various units at Fraunhofer IZI. The objective was to develop innovative technologies for collecting and characterizing circulating tumor cells, to support the development of significantly improved tumor diagnostics and innovative therapies.

  • Illumina sequencing platform Hiseq 2500 (Ultra-High-Throughput Sequencing System)
  • Illumina sequencing platform Miseq
  • Microlab STARlet (Hamilton, automatically prepares samples for sequencing)
  • Qiacube (semi-automated extraction and purification of nucleic acids)
  • Microarray scanner (Agilent, Affymetrix)
  • Perkin Elmer Labchip GX (to determine the quality of nucleic acids, can be integrated into automation)
  • Perkin Elmer Labchip DS (to quantify nucleic acids, can be integrated into automation)
  • Agilent Bioanalyzer (to determine the quality of nucleic acids and proteins)
  • Nanodrop (to quantify nucleic acids)
  • Qubit 2.0 (extremely sensitive fluorometric quantification of specific nucleic acids)
  • Covaris M220 Focused-ultrasonicator™ (nucleic acid shearing, active paraffin removal from FFPE tissues)

  • Hackermüller J, Reiche K, Otto C, Hösler N, Blumert C, Brocke-Heidrich K, Böhlig L, Nitsche A, Kasack K, Ahnert P, Krupp W, Engeland K, Stadler PF, Horn F. Cell cycle, oncogenic and tumor suppressor pathways regulate numerous long and macro non-protein-coding RNAs. Genome Biology. 2014 Mar 4;15(3):R48. DOI http://dx.doi.org/10.1186/gb-2014-15-3-r48
  • Blumert C, Kalkhof S, Brocke-Heidrich K, Kohajda T, von Bergen M, Horn F. Analysis of the STAT3 interactome using in-situ biotinylation and SILAC. J Proteomics. 2013 Dec 6;94:370-86. DOI dx.doi.org/10.1016/j.jprot.2013.08.021
  • Bauer K, Kretzschmar AK, Cvijic H, Blumert C, Löffler D, Brocke-Heidrich K, Schiene-Fischer C, Fischer G, Sinz A, Clevenger CV, Horn F. Cyclophilins contribute to Stat3 signaling and survival of multiple myeloma cells. Oncogene. 2009 Aug 6;28(31):2784-95. DOI dx.doi.org/10.1038/onc.2009.142
  • Cvijic H, Bauer K, Löffler D, Pfeifer G, Blumert C, Kretzschmar AK, Henze C, Brocke-Heidrich K, Horn F. Co-activator SRC-1 is dispensable for transcriptional control by STAT3. Biochem J. 2009 Apr 28;420(1):123-32. DOI dx.doi.org/10.1042/BJ20081989
  • Löffler D, Brocke-Heidrich K, Pfeifer G, Stocsits C, Hackermüller J, Kretzschmar AK, Burger R, Gramatzki M, Blumert C, Bauer K, Cvijic H, Ullmann AK, Stadler PF, Horn F. Interleukin-6 dependent survival of multiple myeloma cells involves the Stat3-mediated induction of microRNA-21 through a highly conserved enhancer. Blood. 2007 Aug 15;110(4):1330-3.