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Richard Bach

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Label-free and amplification-free microRNA-124 detection in human cells

MicroRNAs (miRNAs) are a novel class of evolutionary conserved single-stranded RNAs, which have an important role in the regulation of gene expression at the posttranscriptional level. These short non-coding RNAs (approximately 23 nucleotides long) were discovered in 1993 1 and since that time there have been found 24 521 entries from over 200 species leading to production of more than 30 000 mature miRNAs (June 2013, miRBase.org).

MiRNA biogenesis is a multilevel process involving many enzymes and proteins, hence its regulation is quite different from the previously described regulators of gene expression. Within the canonical model of biogenesis, genes for miRNAs contain their own promoters and are transcribed by RNA polymerase II into primary transcripts (pri-miRNA) 2, 3. Thus unique way of transcription predestines them to have unique properties. An effect of miRNAs is most often based on the binding to the untranslated region (3’UTR) of target mRNA causing degradation (or inhibition) of target mRNA. It is not surprising that they influence numerous cellular processes such as proliferation, differentiation, apoptosis, metastases, angiogenesis, and immune response 4, 5, of these many are connected with diseases including tumor ones. It has been found that miRNAs may have a different expression pattern in a patient with a tumor disease in comparison to healthy subjects, whereas many miRNAs are specific for a given type of cancer 6. Recently, many studies have shown that microRNA-124 (miR-124), usually expressed in developing nervous system, is downregulated in several types of cancers like breast cancer 7, 8, hepatocellular carcinoma 9, lymphoblastic leukemia 10 and prostate cancer 11. MiR-124 also contributes to neural differentiation of neurons 12, regulates proliferation 13 and gastrulation of stem cells 14.

The detection and quantification of miRNAs is very important for the gene expression profiling, however, there are several limitations of miRNAs detection such as their short length and tissue-specific occurrence. Basic methods used for detection are northern blotting, real-time reverse transcription PCR (RT-qPCR), in situ hybridization (ISH) and micro-RNA arrays 15-22. These methods require labelling (radioactive, fluorescent), amplification and/or enzymatic catalysis. With the exception of the RT-qPCR none of these techniques is quantitative. Besides these methods, electrochemical methods can be also used 23. From the electrochemical methods, those detecting reduction of nucleic acids bases on mercury electrodes belong to the most sensitive ones. Palecek was the first who used modern oscillographic polarography for successful detection of redox DNA signals 23, 24. Since then, the attention is paid attention to various electrochemical methods including linear sweep and cyclic polarography/voltammetry (elimination polarography/voltammetry), differential pulse polarography/voltammetry, square wave polarography/voltammetry, AC polarography/voltammetry, and chronopotentiometry for analysis of DNA 23, 25. Square wave voltammetry (SWV) is one of the most sensitive electrochemical methods for determination of oligonucleotides (ODNs) 26. SWV offers background suppression combined with the effectiveness of differential pulsed voltammetry (DPV), slightly greater sensitivity than that of DPV, much faster scan rates, and applicability to a wider range of electrode materials and systems. The most reproducible behavior and the lowest detection limits are generally found at mercury surfaces 23.

Práce je spojená s projektem GACR P102/11/1068 NanoBioTeCell.

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