The Ribolution Project: Automated Screening and Validation for RNA-Biomarkers

  • Automated platform for qPCR based validation of RNA-biomarkersAutomated platform for qPCR based validation of RNA-biomarkers
  • Automated platform for qPCR based validation of RNA-biomarkers

The Ribolution project: Demographic change is a major challenge for society, and also for the funding of the public health system. At present, it is the lack of diagnostic tools that impedes the accurate and cost effective analysis of a disease with a view to its stage, type and severity. With the Ribolution project we present a new innovative approach to automated biomarker screening. Through miniaturization and flexible hardware solutions the complexity and expenditure for screening and validation can be reduced drastically.

Modern methods of molecular diagnostics identify solutions that may revolutionize our health care system and the way we deal with diseases. The focus of attention here is on concentrations of specific RNA molecules found in blood, urine or tissue - the so-called biomarkers - which can be used for the early diagnosis and prognosis of diseases. This approach allows both for more sophisticated forms of treatment tailored to the needs of the patient and for a reduction of costs.

No wonder the demand for biomarkers for specific diseases is high and growing [1]. The process of investigating and validating appropriate indicators, however, is very costly and requires significant experimental effort [2]. Most research projects are faced with insurmountable logistical and financial problems when trying to systematically validate the potential biomarkers on the basis of large patient cohorts. The current market demand for biomarkers can therefore not be met for most diseases.

The Ribolution project [3] involves an interdisciplinary consortium of five Fraunhofer institutes (IPA, IZI, IGB, ITEM, FIT), facing up to the challenge of establishing an efficient and intelligent biomarker screening process. In close collaboration with industry (Glaxosmithkline) and several German universities (Leipzig, Dresden), Fraunhofer explores new territory in integrated biomarker screening. The project is funded by the Fraunhofer Zukunftsstiftung (Fraunhofer Future Foundation).

Next-Generation Biomarker Screening
The consortium has established a multi-stage development pipeline to identify and validate biomarkers. The central pillars of the development process are quality, performance and costs.

The development pipeline is the first to fully address all steps from taking blood or tissue samples to the validation and marketing of the biomarkers.

By focusing on a class of RNA that so far has rarely been considered in screening, the so-called non-coding RNA (ncRNA), the project provides extensive opportunities for application and attractive links for SMEs to the diagnostic market. Due to its regulatory function in the organism, ncRNA is a promising target for a variety of diseases. Resolving the complexity arising from the systematic development of ncRNA-based biomarkers is only possible within reasonable time and cost limits if the methods and automation solutions developed in Ribolution are applied. In the current project, this is demonstrated on socially relevant diseases such as prostate cancer, COPD, and chronic pneumonia.

In a first screening phase, a small and well-characterized patient cohort is analyzed using next-generation sequencing methods. By comparing healthy and diseased donors with the aid of efficient bioinformatics methods, potential sequences for biomarker candidates are identified.

Both the quality of the RNA samples and their full traceability have a critical effect on the quality of the sequencing and the characterization of the biomarker candidates. The project takes account of this by using improved methods for the extraction of RNA from tissue, blood or urine. For instance, all steps in the extraction and purification of samples were analyzed in close consultation with staff from hospital, molecular biological analysis and process automation, resulting in the establishment of new quality standards.

The screening phase is followed by the validation of the candidate biomarkers characterized before. Quantitative real-time PCR is used to test the identified candidates on large cohorts of up to 500 patients for each disease. Accordingly, about 500,000 PCR assay points arise from the validation of the diseases and cohorts addressed by Ribolution.

Based on the I-Dot technology [6], the project applies qPCR-based diagnostic methods on new scales. By miniaturizing the assays to the sub-microliter scale and precisely dispensing individual media in volumes of few nanoliters, new and less costly ways of automation are enabled. The advantages over conventional screening are the reduction of consumable costs per assay point (Fig. 1) and the reduced need for expensive RNA samples. The significant reduction of the volumes of the PCR assays allows for an increased parallelization of assays (1536 assay points per microtiter plate).

For the first time, consistently SiLA compatible devices and device drivers for integrating and networking the devices [7] had been used. SiLA defines a standardized communication interface, enabling the quick and easy networking of system components. The networked hardware and the control and scheduling software LACS (Laboratory Automation Control Suite) are combined into a development tool. The unique combination of SiLA and LACS helps to keep the lab infrastructure smart and lean for a screening project such as shown here, without neglecting process reliability and performance. In addition, it is possible to reduce service times for the repair and integration of devices, thus creating ample room for innovation for future automation solutions.

Ribolution Screening Center
The work and developments undertaken here enable the biological researchers and the pharmaceutical industry for the first time to develop new biomarkers with reasonable effort and cost. The volume of RNA required per candidate marker in the screening process can be reduced by 98% by process miniaturization and the possibility to dispense in sizes of single nanoliters (Fig. 2). It is expected that the reduced need for RNA, along with the screening pipeline described above, will make it much easier to access the valuable cohorts and RNA libraries. The time advantage over conventional solutions can be directly translated into scientific and economic advantages and a competitive edge in development.

To make the here shown technologies directly and easily accessible for new project partners and also to include new diseases, a Screening Center is to be founded in a second funding phase. This center will combine the developed technologies and offer numerous interfaces to SMEs as well as to possible licensees and commercial exploitation partners. The authors are convinced that this project will help to strengthen and expand the leading position of Germany as a research and business location for molecular and personalized diagnostics.

References
[1] Frost & Sullivan, Advances in Biomarkers, D19A-01, p. 01, 2009
[2] Frost & Sullivan, Advances in Biomarkers, D19A-01, p. 32, 2009
[3] http://www.ribolution.org, 11/2013
[4] J. S. Mattick: Non-coding RNA. In: Human molecular genetics. Vol. 15 Spec No 1, April 2006
[5] BIOforum Europe, Git Verlag, Vol13, July 2009
[6] i-doT-System - Bioproduktion am Fraunhofer IPA; http://www.bioproduktion.com/idoT.html, 11/2013
[7] SiLA Rapid Integration | Standardization in Lab Automation. http://www.sila-standard.org; 11/2013

Corresponding author:
Dipl.-Phys. Mario Bott
Tel.: +49 711 970-1029
Fax: +49 711 970-1005

 

Contact

Fraunhofer - Institut für Produktionstechnik und Automatisierung IPA
Nobelstr. 12
70569 Stuttgart
Germany
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Telefax: +49 711 970-1399

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