Research
Topics
Methodology and Quality Controls
We are very active in the areas of new method development and implementation. In the past, the laboratory pioneered single cell RT-qPCR expression profiling, multidimensional expression profiling, intracellular expression profiling by qPCR, developed a new method for miRNA analysis and introduced several tools and protocols for quality control of RT-qPCR experiments. Our group leader Prof. Mikael Kubista, was actually among the pioneers who developed real-time PCR. Currently the main focus of our lab is the investigation of gene expression using a combination of up-to date techniques such as single cell expression profiling and RNA-Seq, with the aim to measure both coding mRNA and also non-coding RNA (miRNA, lncRNA etc.).
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 :
- 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.
- Tutorial: Guidelines for Single-Cell RT-qPCR. Zucha D, Kubista M, Valihrach L. Cells. 2021; 10(10):2607. https://doi.org/10.3390/cells10102607.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
Developmental Biology
Our group uses the African clawed frog model for three main projects – asymmetric localization of biomolecules during oogenesis and early development; regulation of wound healing and regeneration; and study of role of nitric oxide during early embryogenesis. We developed techniques for spatial and temporal expression profiling using RT-qPCR and more recently RNA-Seq, and we have combined them with functional experiments using microinjections and phenotype analysis such as in situ hybridization and imaging.
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 :
- 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.
- 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.
- 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.
- 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.
Grants
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AV Mobility+: AS-25-03 |
Deciphering ROS Signaling in Tissue Regeneration and Cancer Progression: A Collaborative Approach Using Advanced Redox Profiling Techniques |
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Multi-omics platform for the search for biological correlates of disease and the development of new diagnostic, preventive and therapeutic approaches |
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Comparative transcriptomic analysis of regeneration in head and neck cancer as a new strategy to search cancer prognostic markers. 2024–2027. |
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A humanized organoid-based platform for stroke modelling and mRNA-based therapeutic intervention. 2024–2026. |
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Regeneration initiation cells - a new player in healing and regeneration. 2024–2026. |
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Constructing the Developmental Atlas of Alzheimer’s Disease using Cerebral Organoids and Single-Cell Transcriptomics. 2024-2026. |
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NU22-06-00643 |
Receptivity of endometrial cavity in patients with fertility limiting diseases of pelvic organs: prospective clinical study (REAdME). 2023-2026. |
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GA23-05327S |
Astrocyte-microglia communication as a target for stroke therapy. 2023-2025. |
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GA23-06269S |
Disturbed regulation of mTOR signaling in glial cells following cerebral ischemia. 2023-2025. |
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GA22-10660S |
The role of Meis transcription factors in mesenchymal condensations during formation of the cranium. 2022-2024. |
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Circulating microRNAs as biomarkers for evaluating the severity of acute spinal cord injury. 2021-2024. |
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QK21010030 |
GIPTSAL - Globalization, modern technologies and climate change provide both new opportunities and hazards for salmonid breeding management. 2021-2025. |
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GA20-23836S |
Sturgeon as a unique model for evolutionary transition from holoblastic to meroblastic cleavage pattern, and for endoderm development in vertebrates. 2020-2022. |
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ALEXANDER - The astrocyte nanofilament system in Alexander disease – from molecules to function, uncovering new leads for therapy. 2020-2022. |
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The role of glial TRPV4 channels in brain edema formation and post-ischemic regeneration. 2020-2022. |
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Glial cells- the key players in the progression of amyotrophic lateral sclerosis. 2019-2021. |
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| GA19-11313S | Interspecies comparison of RNA localization within oocytes to elucidate regulation of early development and asymmetric cell division. 2019-2021. |
| GA18-21942S | MicroRNA in central nervous system injury: potential roles and therapeutic implications. 2018-2020. |
| GA17-24441S | Molecular mechanisms of proliferation during tumor progression and wound healing studied by profiling on single cell level. 2017-2019. |
| GA17-04034S | Principal signaling pathways regulating NG2 glia proliferation and differentiation following brain injuries. 2017-2019. |
| GA16-07500S | Distribution of biomolecules studied on single cell and subcellular levels in Xenopus early stages to reveal mechanisms of asymmetric cell division. 2016-2018. |
| GA16-10214S | Age-related changes in brain diffusivity, extracellular matrix composition and glial physiology - impact on pathogenesis of ischemia. 2016-2018. |
| GA15-08239S | The diagnostic and prognostic role of microRNA signature in rectal cancer. 2015-2017. |
| LH15074 | Elucidating the mechanism of asymmetric cell division using Xenopus oocytes as model system. 2015-2017. |
| GA13-02154S | Gene expression profiling and functional characterization of glial cell subpopulations following ischemic brain injury. 2013-2016. |
| LK21305 | NO signaling during early embryogenesis and regeneration. 2013-2015. |
| GAP304/12/1585 | Molecular DNA repair characteristics in colorectal tumor tissue. 2012-2015. |
| CZ.1.07/2.3.00/30.0045 | Biotechnological expert in structural biology and gene expression. 2012-2015. |
| ME10052 | Cellular Expression Signatures in Idiopathic Pulmonary Fibrosis. 2010-2012. |
| GAP303/10/1338 | Cell volume regulation in glial cells during brain ischemia/reperfusion. 2010-2012. |
| HEALTH-F5-2008-222916 | Standardisation and improvement of generic pre-analytical tools and procedures for in vitro diagnostics. 2009-2012. |
| NS9976 | Gene expression profiling in cancer circulating cells (CTCs) in breast carcinoma patients - a tool for early metastasis detection and therapy individualisation. 2009-2011. |
| IAA500970904 |
MicroRNA expression during early development of Xenopus laevis and mRNA degradation. 2009-2011. |
Publications
You can look on this page for more info.
Laboratory of Gene Expression
Institute of Biotechnology CAS, v.v.i.
Prumyslova 595
252 50 Vestec
Czech Republic
Phone: +420 325 873 748, +420 325 873 747
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