Ongoing research projects and our grants

Single-cell and sub cell qPCR

In collaboration with TATAA Biocenter, we are developing methods to study the expression of genes in individual cells, and to correlate the expression to physiologic cell properties such as membrane potential as measured by patch clamp technique on the same cell. We also study heterogeneity on cellular level, and have found that expression of certain genes can differ several orders of magnitude between cells in the same seemingly homogeneous population (Genome Research 15, 1388-1392, 2005, Nature Reviews Genetics 6, 1, 2006). In particular, we discovered that expression of genes in individual cell varies according to the log normal distribution.

PROJECT: SINGLE CELL GENE EXPRESSION PROFILING OF ASTROCYTES

In neurotrauma, brain ischemia or neurodegenerative diseases, astrocytes become reactive (which is known as reactive gliosis) At an early stage after neurotrauma, reactive astrocytes have a neuroprotective effect; at a later stage, they facilitate the formation of posttraumatic glial scars and inhibit CNS regeneration. This is accompanied by an altered expression of many genes. We propose that reactive astrocytes can be the future target for the therapeutic strategies promoting regeneration and plasticity in the brain and spinal cord in various disease conditions. To describe and to better understand these processes on gene expression level we make expression profiles of astrocytes on single cell level using real-time PCR (qPCR). Our unique high-throughput qPCR platform allows us to study up 100 genes per cell, using our optimized protocols.

PROJECT: CELL VOLUME REGULATION IN GLIAL CELLS DURING BRAIN ISCHEMIA

The aim of this study is to identify the major ion channels or transporters participating in astrocyte and NG2 glia swelling and volume regulation during ischemia or hypotonic stress in brain slices. Changes in volume of astrocytes given by ischemia results brain edema and a full understanding of astrocyte and NG2 glia responses to ischemic insult and their underlying mechanisms is important for the future development of glia-oriented therapeutic strategies. We collaborate with Department of Cell Neurophysiology on this project. Our part of the work is gene expression profiling ion channel on single cell level. These cell are directly collected into lysis buffer and it is performed the reverse transcription from one cell. Then the preamplification is performed and qPCR. We used Biomark platform for these measurements.

Expression profiling

In collaboration with MultiD Analyses we develop methods for multivariate real-time PCR expression profiling. These methods are particularly powerful for the classification of genes and samples with similar expression patterns, and they are useful for applications from the identification of expression pathways to classification of diseases based on expression profiles.The great accuracy, extreme sensitivity and wide dynamic range of real-time PCR technology has opened for highly accurate expression profiling studies of complex biological phenomena including embryonic development and complex diseases. In particular multidimensional studies, where several parameters are varied, such as measuring the expression of genes over time and space (or drug load) are expected to be exceedingly valuable and important. In this project multivariate and multiway methods will be adapted and further developed for the analysis of multidimensional real-time PCR gene expression profiling data. The work requires mathematical and statistical skills, but will also have experimental parts.

PROJECT: A SPATIO - TEPMORAL MAPPING OF GENE EXPRESSION DURING DEVELOPMENT OF MAMMAL ORGAN USING LASER-MICRODISECTION ASSISTED qPCR TOMOGRAPHY

Recent methods used in developmental biology, such as ISH, whole mount ISH, microarrays or SAGE, often give limited information on gene expression in the context of multiple genes expression and spatialy distribution along the whole studied subject. Limitations come from a low level of detection, sensitivity, dynamic range and also high cost of these methods. The aim of our project is studying the development of a mouse molar using our newly developed qPCR tomography technology combined with laser-cutting microdissection (LCM). Mouse embryos are sacrified among 12-16 days of their development. This interval includes epithelial-mesenchymal interactions which control patterning and morphogenesis of the molar. We start mapping these processes at early stages of tooth forming, the map will also include distribution of signaling centers till the final phase of the differentiation of odontogenic cells. 25-30 developmental or structure genes were selected for this study. Our goal is to generate the first 3-dimensional spatiotemporal expression map of a developing tooth. BioMark platform is used for high-throughput qPCR analysis.

Immuno-qPCR

In many cases expression of proteins is more informative than gene expression profiles. We have combined the sensitivity and accuracy of QPCR with the specificity of immunoassays in immuno-Q-PCR (J. Immun. Meth. 304, 107-116, 2005). Binding the protein by two specific antibodies, one of which is tagged with an oligonucleotide, we can, after careful washing, determine the amount of target protein by amplifying the DNA. Immuno-Q-PCR can be performed in most conventional standard Q-PCR instrument.

Standardisation of generic pre-analytical tools and procedures for in vitro diagnostics

In vitro diagnostics have allowed a great deal of progress in medicine but are limited by two factors: (a) the lack of guidelines in collection, handling, stabilisation and storage of biosamples which limits the reproducibility of subsequent diagnoses, and (b) its scale is restrained to the cellular level. To address this first point, this IP, SPIDIA, built of clinicians, academics, tool and assay developers, aims at developing quality guidelines for molecular in vitro diagnostics and to standardise the pre-analytical workflow in related procedures. Regarding the second point, SPIDIA aims at developing modern pre-analytical tools for diagnostics improving the stabilisation,handling and study of free biomolecules within blood, plasma, serum, tissues and tumours. Recent discoveries have revealed that RNA, DNA or proteins, released from pathological sites, like tumour cells or Alzheimer's disease (AD) brain lesions, into the blood or as a secondary blood based response to the disease can serve as biomarkers for early and reliable molecular diagnosis of such debilitating diseases. Further discoveries have shown that the cellular profiles of these molecules and structures in clinical samples can change during transport and storage thus making clinical assay results and pharmaceutical research unreliable or even impossible. It will therefore be a decisive prerequisite for future and current diagnostic assays to develop standards and new technologies, tools and devices that eliminate the human error in the pre-analytical steps of in vitro diagnostics. At this crucial moment in the development of molecular diagnostics, SPIDIA proposes an IP that reunites 7 private research companies (including 4 SMEs), 1private research institute and 6 public research organisms, including universities, hospitals and biobanks, one management SME and an official European Standards Organisation. This strong consortium is balanced and empowered to maximise the impacts of in vitro diagnostics on human health.

IBT tasks:

• Identification and Validation of biomarkers to monitor changes in clinical sample materials
• Identify RNA biomarkers affected by pre-analytical variation in blood and tissue samples
• Study of stabilisation components used for fine needle aspirates
• Evaluation of the plasma/serum stabilisation and concentration technology
• Evaluation of the applicability of the new pre-analytical system in biomarker discovery programs
• Training, dissemination and exploitation of results

New dyes for qPCR

We are developing and evaluating new dyes for real-time PCR applications. Our dyes are designed to bind in the DNA minor groove, which makes them more selective for double-stranded DNA than, for example, SYBRGreen. The dyes are designed with different colours and can be combined with probes for quality control (Biotechniques 40, 315-319, 2006). The dyes are also excellent for high resolution melt applications.

Molecular basis of intra-cellular mRNA gradients

Even the first cell - the oocyte - is asymmetric. Within the cell gradients of organelles, proteins and mRNA are found, such that when the first cell divides the two daughter cells are different. Asymmetric cell divisions continue throughout early development giving rise to a complex organism. To study intracellular mRNA gradients our group has developed sub-cellular real-time PCR. Using the African claw frog Xenopus laevis as model system we freeze its gigantic eggs, slice them and measure mRNA distribution within the oocyte. In this project we will study the gradients in more detail, and in particular how they change upon fertilization. In an extension of the study we will set up an in vitro selection system to identify base sequences that are important for the formation of mRNA gradients. This will help to elucidate the mechanism behind intracellular mRNA gradient formation and can also reveal new molecular causes to human diseases. The work is mainly experimental.

Novel technologies for nucleic acid detection

Today expression of genes is measured mainly by first copying the mRNA into cDNA which then is quantified by means of real-time PCR or some other technique based on PCR amplification or hybridization to microarrays. The techniques are excellent for studies of large samples, but are difficult to apply on limited sample amounts and single cell studies. In this project we develop novel and more sensitive methods for nucleic acid detection and quantification.

Methods to analyze multidimensional gene expression data

The expression of genes reflects the genetic predisposition of an individual as well as her current physiologic state. This makes expression profiles powerful indicators of individual's state of health. As techniques for genetic profiling are being refined, they are rapidly gaining popularity in biomedical research, pharmaceutical development, molecular diagnostics and disease monitoring of treated patients. The most powerful technique for gene expression profiling is realtime PCR, by which amounts of mRNA in biological samples can be measured with unprecedented accuracy and sensitivity. In collaboration with professor Richard Breretron at Univeristy of Bristol, Dr Jan Paces at Institute of Molecular Genetics, Prague, Dr José Manuel Andrade at University of A Coruna, La Coruna, and MultiD Analyses AB we develop methods based on support vector machines and neural networks to classify realtime PCR gene expression data.

Cancer research

PROJECT: GENE EXPRESSION PROFILING IN CIRCULATING TUMOR CELLS (CTCs) IN BREAST CANCER PATIENTS - A TOOL FOR EARLY METASTASIS DETECTION AND THERAPHY INDIVIDUALIZATION

Despite recent advances in the diagnostics and therapy of breast cancer many patients with primary breast cancer, even when diagnosed at early stage, eventually suffer a relapse of the disease and ultimately die. A systemic relapse of breast cancer occurs after curative operation in 20-40% of the patients, even for those with a tumor size less than 2 cm in diameter independently of nodal status. It is clear that the hematogenous route is a significant route for tumor dissemination. The presence of circulating tumor cells (CTCs) in the blood indicates disease progression. Their abundance reflects a relapse or metastating process, since CTCs survive only some 24 hours in the circulation. The main goal of the project is to monitor disease progress in breast cancer patient by measuring the gene expression of disease marker (MUC-1, EpCAM, HER2/neu and other potentional oncomarkers) in CTCs. Based on the number of CTCs in blood circulation and the level of biomarker gene expression we monitor the dynamics of the disease and the effect of the chemotherapy the patient is given, which allow us to optimize the treatment for every individual. This is expected to be one of the most important progresses in the therapy of cancer – the personalized therapy.