Research 

Single-Cell Expression Profiling in Research and Diagnostics

The project develops tools for the spatio-temporal analysis of the expression of genes with the help of RNA-Seq and high-throughput RT-qPCR at the tissue, unicellular and sub-cellular level, including multidimensional data analysis. We aim to develop more powerful methods for diagnostics, disease monitoring and progression of complex human diseases, such as cancer, based on the prevalence and phenotypic profiling of rare disease determining cells.

Quality control tools

Single-cell gene expression profiling is revolutionizing the exploration of expression pathways, networks and biomolecular interactions. This field is currently in rapid development, both theoretically as well as experimentally. To ensure high quality of data, we continuously develop new approaches for quality control and data analysis. In the past, we presented a method for the effective RNA extraction from single cells, a method to control gDNA contamination or the quality of RNA, described the advantages in measuring bulk samples in single cell experiments and also participated in establishing guidelines for gene expression profiling (MIQE guidelines).

Novel method for highly accurate miRNA quantification

MicroRNAs are a class of small non-coding RNAs that serve as important regulators of gene expression at the post-transcriptional level. They are stable in body fluids and pose a great potential to serve as biomarkers. We developed a highly specific, sensitive and cost-effective system to quantify miRNA expression based on two-step RT-qPCR with SYBR-green detection chemistry called Two-tailed RT-qPCR. It takes advantage of novel, target-specific primers for reverse transcription composed of two hemiprobes complementary to two different parts of the targeted miRNA, connected by a hairpin structure. The introduction of a second probe ensures high sensitivity and enables discrimination of highly homologous miRNAs irrespective of the position of the mismatched nucleotide. Currently, the method is employed in various projects, including the role of miRNA in spinal cord injury and also in the progression of viral infection.

Selected publications :

1. Small RNA-Sequencing for Analysis of Circulating miRNAs: Benchmark Study. Androvic P, Benesova S, Rohlova E, Kubista M, Valihrach L. J Mol Diagn. 2022 Jan 23:S1525-1578(22)00011-3. doi: 10.1016/j.jmoldx.2021.12.006.
2. Tutorial: Guidelines for Single-Cell RT-qPCR. Zucha D, Kubista M, Valihrach L. Cells. 2021; 10(10):2607. https://doi.org/10.3390/cells10102607.
3. Small RNA-Sequencing: Approaches and Considerations for miRNA Analysis. Benesova S, Kubista M, Valihrach L. Diagnostics (Basel). 2021 May 27;11(6):964. doi: 10.3390/diagnostics11060964.
4. Performance Comparison of Reverse Transcriptases for Single-Cell Studies. Zucha D, Androvic P, Kubista M, Valihrach L. Clin Chem. 2019 Nov 7. pii: clinchem.2019.307835. doi: 10.1373/clinchem.2019.307835.
5. Multicenter Evaluation of Circulating Plasma MicroRNA Extraction Technologies for the Development of Clinically Feasible Reverse Transcription Quantitative PCR and Next-Generation Sequencing Analytical Work Flows. CANCER-ID consortium. Clin Chem. 2019 Sep;65(9):1132-1140. doi: 10.1373/clinchem.2019.303271.
6. Two-tailed RT-qPCR panel for quality control of circulating microRNA studies. Androvic P, Romanyuk N, Urdzikova-Machova L, Rohlova E, Kubista M, Valihrach L. Sci Rep. 2019 Mar 12;9(1):4255. doi: 10.1038/s41598-019-40513-w.
7. Platforms for Single-Cell Collection and Analysis. Valihrach L, Androvic P, Kubista M. Int J Mol Sci. 2018 Mar 11;19(3). pii: E807. doi: 10.3390/ijms19030807.
8. Two-tailed RT-qPCR: a novel method for highly accurate miRNA quantification. Androvic P, Valihrach L, Elling J, Sjoback R, Kubista M. Nucleic Acids Res. 2017 Sep 6;45(15):e144. doi: 10.1093/nar/gkx588.

Gene expression profiling and characterization of glial cell subpopulations following ischemic brain injury

Stroke is the third leading cause of death in industrialized countries and leads to serious, long-term disability for survivors. Glial cells, comprising astrocytes, oligodendrocytes, microglia and NG2 glia, represent homeostatic systems that regulate all aspects of CNS function and may have beneficial as well as detrimental effects on the outcome of ischemic injury. The mechanisms involved in reactive glial functions are still not well understood and are studied within this group of projects. We are interested in the role astrocytes and NG2 cells in pathophysiology of ischemia, we study their dynamics, differentiation and proliferation capacity, age-dependent changes in their function and structure, role of specific regulation pathways and proteins, or similar expression patterns in different types of CNS injury. 

Glial cells - the key players in the progression of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that affects the upper and lower motor neurons and results in progressive paralysis, with a mortality within 2-5 years after the onset of disease. Although ALS is characterized by the selective death of motor neurons, glial cells and their communication processes play a key role in its pathology. While the importance of astrocytes and microglia in the development of ALS has been already described, the role of other glial cell subtypes such as oligodendrocytes and NG2 glia remains unclear. The aim of this project is to identify the changes in astrocytes, microglia and NG2 glia/oligodendrocytes in ALS progression at the level of gene and protein expression. Furthermore, we aim to clarify the changes in their functional properties, especially the homeostatic functions of astrocytes, and the role of NG2 glia in oligodendrogenesis and myelination of axons.

MicroRNA in central nervous system injury: potential roles and therapeutic implications

Central nervous system (CNS) injury triggers a multitude of pathophysiological events that are tightly regulated by expression of specific genes. Recent studies have demonstrated, that microRNAs (miRNAs) – short non-coding RNAs that modulate protein expression levels by antagonizing mRNA – are dysregulated following different types of CNS injuries. This observation has sparked a significant amount of attention into the potential use of microRNAs as therapeutic targets. To understand the mechanisms underlying gene alterations following spinal cord injury and ischemic stroke, we examine temporal expression of miRNAs and mRNAs in the nervous tissue in mice and rat models. The most promising miRNA-mRNA regulatory networks are validated in vitro and in vivo using a combination of tools for RNA, protein and functional analysis to verify the true biological relevance of our findings.

Single-cell transcriptomic analysis of Alexander disease

Alexander disease (AxD) is a rare genetic brain disease with no available treatment. AxD patients show white matter degeneration and extensive loss of brain tissue. Symptoms include mental retardation, epilepsy, muscle stiffness, and AxD leads to death. AxD is caused by mutations in GFAP, an astrocyte intermediate filament (nanofilament) protein. The disease initiates in astrocytes, cells that control many neuronal functions and homeostatic mechanisms of the brain. However, the molecular and cellular circuitry underlying AxD is unknown. Within the project, we investigate the cellular and molecular mechanisms leading to AxD and identify targets for potential treatment strategies. We use wide spectrum of cutting-edge technologies (including single-cell transcriptomics) and unique biological models (patient-derived iPS cells, cerebral organoids or AxD killifish model).

Selected publications:

1. Transient astrocyte-like NG2 glia subpopulation emerges solely following permanent brain ischemia. Kirdajova D, Valihrach L, Valny M, Kriska J, Krocianova D, Benesova S, Abaffy P, Zucha D, Klassen R, Kolenicova D, Honsa P, Kubista M, Anderova M. Glia. 2021 Jul 27. doi: 10.1002/glia.24064.
2. Decoding the transcriptional response to ischemic stroke in young and aged mouse brain. Androvic P, Belov Kirdajova D, Tureckova J, Zucha D, Rohlova E, Abaffy P, Kriska J, Anderova M, Kubista M, Valihrach L. Cell Rep. 2020 Jun 16;31(11):107777. doi: 10.1016/j.celrep.2020.107777.
3. Circulating miRNA analysis for cancer diagnostics and therapy. Valihrach L, Androvic P, Kubista M. Mol Aspects Med. 2019 Oct 18:100825. doi: 10.1016/j.mam.2019.10.002.
4. A single-cell analysis reveals multiple roles of oligodendroglial lineage cells during post-ischemic regeneration. Valny M, Honsa P, Waloschkova E, Matuskova H, Kriska J, Kirdajova D, Androvic P, Valihrach L, Kubista M, Anderova M. Glia. 2018 May;66(5):1068-1081. doi: 10.1002/glia.23301.

Localization of biomolecules leading to asymmetric cell division

Asymmetrical localization of RNA and proteins is the main factor behind cell specification and differentiation. Early development is a great model, because asymmetry can be observed even in oocytes and eggs.  We use primarily the Xenopus laevis model to identify groups of localized RNAs and proteins, and rely on Bioinformatic analysis to assist with the determination of localization motifs and elements in the RNA molecules. We study all main developmental axes: both the oocyte and egg to study animal-vegetal axis (ectoderm – endoderm); 2-cell stage embryos for left-right asymmetry; and 4-cell stage embryos and older for dorsal-ventral axis. Recently, our collaboration with the laboratory of Prof. Dovichi in USA has been successful in also measuring protein asymmetrical profiles and their uneven distributions within the blastomeres of developing embryos. Our research in this field has led us to the discovery of a new group of transcripts with animal localization and also the identification of several new localization motifs within the vegetally localized transcripts. In addition to Bioinformatics, we also test validity of localization motifs using functional experiments. In 2018, we started new collaborations using fish and mammalian egg models.

The role of nitric oxide during development

The nitric oxide molecule is used extensively in various biological systems. Our research on the role of this molecule has shown that it is essential for head development and skin formation. We have also observed that this molecule is strongly produced in ciliated and secretory cells and it is required during the initial steps of epidermis formation. Our lab also study the role of nitric oxide during wound healing and regeneration. We found that nitric oxide is strongly produced in just a few minutes after injury and that it has a key role in gene expression activation.

Gene expression during wound healing and regeneration

The main aim of our project is to uncover the molecular mechanisms and signalling networks regulating embryonic wound healing and regeneration. It utilizes the unique combination of state-of-the-art transcriptomic and proteomic approaches, followed by candidate selection using bioinformatic analysis and functional analysis of candidates during wound healing and regeneration. Our results can be used for the identification of therapeutic targets to improve the treatment of chronic wounds and burns. Embryonic healing and regeneration are both fascinating biological processes and amphibians including Xenopus embryos are among the most efficient model species for their study. We have performed large-scale analysis of healing using transcriptomic and proteomic analyses and identified several signalling pathways and genes, which we are currently studying in more details. Recently we have developed an efficient workflow for single cell expression analysis and have used it to determine cell specific regeneration processes at the single cell resolution.

Selected publications :

1. Intracellular expression profiles measured by real-time PCR tomography in the Xenopus laevis oocyte. Sindelka R, Jonák J, Hands R, Bustin SA, Kubista M. Nucleic Acids Res. 2008. 36(2):387-92.
2. The extreme anterior domain is an essential craniofacial organizer acting through Kinin-Kallikrein signaling. Jacox L, Sindelka R, Chen J, Rothman A, Dickinson A, Sive H. Cell Rep. 2014. 8(2):596-609. doi: 10.1016/j.celrep.2014.06.026.
3. Single blastomere expression profiling of Xenopus laevis embryos of 8 to 32-cells reveals developmental asymmetry. Flachsova M, Sindelka R, Kubista M. Sci Rep. 2013;3:2278. doi: 10.1038/srep02278.
4. The role of nitric oxide during embryonic epidermis development of Xenopus laevis. Tomankova S, Abaffy P, Sindelka R. Biol Open. 2017. 6(6):862-871. doi: 10.1242/bio.023739.