• MeSH
• Biomedical Research MeSH
• Databases, Genetic MeSH
• Research Support as Topic MeSH
• Financing, Organized MeSH
• Clinical Medicine MeSH
• Humans MeSH
• Oligonucleotide Array Sequence Analysis MeSH
• Software MeSH
• Gene Expression Profiling MeSH
• Systems Integration MeSH
• Database Management Systems MeSH
• Information Storage and Retrieval MeSH
• Computational Biology MeSH
• Check Tag
• Humans MeSH

Metallothioneins (MTs), low molecular mass cysteine-rich proteins, which are able to bind up to 20 monovalent and up to 7 divalent heavy metal ions are widely studied due to their functions in detoxification of metals, scavenging free radicals and cells protection against the oxidative stress. It was found that the loss of the protective effects of MT leads to an escalation of pathogenic processes and carcinogenesis. The most extensive area is MTs expression for oncological applications, where the information about gene patterns is helpful for the identification biological function, resistance to drugs and creating the correct chemotherapy. In other medical applications the effect of oxidative stress to cell lines exposed to heavy metals and hydrogen peroxide is studied as well as influence of drugs and cytokines on MTs expression and MTs expression in the adipose tissue. The precise detection of low metallothionein concentrations and its isoforms is necessary to understand the connection between quantity and isoforms of MTs to size, localization and type of cancer. This information is necessary for well-timed therapy and increase the chance to survival. Microarray chips appear as good possibility for finding all information about expression of MTs genes and isoforms not only in cancer, but also in other diseases, especially diabetes, obesity, cardiovascular diseases, ageing, osteoporosis, psychiatric disorders and as the effects of toxic drugs and pollutants, which is discussed in this review.

• MeSH
• Humans MeSH
• Metallothionein analysis   genetics   metabolism   MeSH
• Microarray Analysis methods   MeSH
• Neoplasms diagnosis   genetics   metabolism   MeSH
• Oxidative Stress physiology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH

The left and right ventricle originate from distinct parts of the cardiac tube, and several genes are known to be differentially expressed in these compartments. The aims of this study were to determine developmental differences in gene expression between the left and right ventricle, and to assess the effect of altered hemodynamic loading. RNA was extracted from isolated left and right normal chick embryonic ventricles at embryonic day 6, 8, and 10, and from day 8 left atrial ligated hearts with hypoplastic left and dilated right ventricles. cRNA was hybridized to Affymetrix Chicken Genome array according to manufacturer protocols. Microarray analysis identified 302 transcripts that were differentially expressed between the left and right ventricle. Comparative analysis detected 91 genes that were different in left ventricles of ligated hearts compared to age-matched ventricles, while 66 were different in the right ones. A large number of the changes could be interpreted as a delay of normal maturation. The approach described in this study could be used as one of the measures to gauge success of surgical procedures for congenital heart disease and help in determining the optimal time frame for intervention to prevent onset of irreversible changes.

• MeSH
• Hemodynamics MeSH
• Chick Embryo MeSH
• Microarray Analysis MeSH
• Myocardium metabolism   MeSH
• Heart Ventricles embryology   metabolism   MeSH
• Heart Atria embryology   metabolism   MeSH
• Transcriptome MeSH
• Animals MeSH
• Check Tag
• Chick Embryo MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH

V poslední době se v experimentální praxi stále ve větší míře uplatňují metody využívající mikročipové expresní analýzy. Většina expresních analýz v medicínském výzkumu vychází z bioptických vzorků, které obsahují řadu různých typů buněk. Majoritní populace buněk je určující pro celkovou informaci o genové expresi a znemožňuje tak rozlišení specifické genové exprese jednotlivých typů buněk. Mezi metody, které umožňují selekci přesně definovaných populací buněk patří laserová a mechanická mikrodisekce. Současné čipy vyžadují mikrogramová množství značené nukleové kyseliny, přičemž vstupní množství RNA ze selektovaných buněk se může pohybovat pouze v nanogramovém až pikogramovém řádu. Tento problém je možno řešit různými způsoby amplifikace RNA, mezi které patří dvojitá lineární amplifikace RNA nebo metody využívající kombinovanou PCR s linearní amplifikací. V tomto krátkém přehledu poskytujeme jednak informace, se kterými jsme se setkali jednak během naší současné analýzy genové exprese v mikrodisekovaných buňkách mléčné žlázy, a také náš optimalizovaný postup. Celý projekt sestával z časově náročného sběru čerstvě zmražené tkáně, optimalizace přípravy kryofiezů pro mikrodisekci, izolace a amplifikace RNA, hybridizace na mikročipy, analýzy dat a konečně verifikace vybraných výsledků pomocí imunohistochemie. V současné době je vlastní práce v oponentním řízení v zahraničním časopise, a nemůžeme proto poskytnou detailnější informace o získaných výsledcích.

The DNA microarray is a powerful, high throughput technique for assessing gene expression on a systemwide genomic scale. Most expression profiling studies of solid tumors have used biopsy samples containing large numbers of contaminating stromal and other cell types, thereby complicating any precise delineation of gene expression in nontumor versus tumor cell types. Combining microdissection, RNA amplification protocols, microarray technologies and our knowledge of the human genome sequence, it is possible to isolate pure populations of cells or even a single cell and interrogate the expression of thousands of sequences for the purpose of more precisely defining the biology of the tumor cell. In this short overview, we provide informations on selected problems which we had to solve during our microarray analysis of microdissected normal and tumour cells of mammary gland. We provide our optimized procedure as well. The whole project consisted of the long-term collection of snap-frozen tissues, optimalization of staining of cryosections for laser capture microdissection, isolation and amplification of RNA, hybridization onto chips, data analysis and finally verification of the results by immunohistochemistry. Our work is currently reviewed by an international journal and we can’t provide detailed information on particular achievements yet.

• MeSH
• Protein Array Analysis methods   instrumentation   utilization   MeSH
• Gene Expression genetics   MeSH
• Financing, Organized MeSH
• Immunohistochemistry utilization   MeSH
• Lasers utilization   MeSH
• Humans MeSH
• Microdissection methods   instrumentation   utilization   MeSH
• Mammary Glands, Human cytology   MeSH
• Specimen Handling methods   trends   MeSH
• Reverse Transcriptase Polymerase Chain Reaction methods   instrumentation   utilization   MeSH
• RNA diagnostic use   genetics   isolation & purification   MeSH
• Nucleic Acid Amplification Techniques methods   instrumentation   utilization   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Review MeSH

• MeSH
• Molecular Targeted Therapy MeSH
• Molecular Diagnostic Techniques MeSH
• Precision Medicine MeSH
• Medical Oncology MeSH
• Microarray Analysis MeSH
• Research MeSH
• Publication type
• Collected Work MeSH

• Conspectus
• Patologie. Klinická medicína
• NML Fields
• onkologie

Účel přehledu: Metodika velkokapacitních mikropolí (microarray), která obsahují různé alergenové složky fixované na pevné podložce, umožňuje stanovení specifických IgE proti těmto alergenům a přesnější diagnostiku potravinové alergie. V tomto přehledovém článku rozebíráme výsledky, jichž bylo dosud technikou mikropolí dosaženo v diagnostice potravinové alergie. Nové poznatky: Technologie mikropolí obsahujících proteiny nebo glykoproteiny nám umožňuje zjistit profil senzibilizace pacientů s potravinovou alergií. V současné době jsou komerčně dostupné metody, jež nám umožňují stanovit specifické IgE proti 103 alergenním molekulám. V celosvětovém měřítku byla v některých laboratořích tato technika zkoumána a optimalizována pro několik alergenových extraktů a výsledky byly slibné – metodika měla vysokou specificitu a senzitivitu, výsledky navíc dobře korelovaly s již existujícími konvenčními testy. Souhrn: V posledních letech se díky pokroku v molekulární biologii a ve vývoji nových technologií pro výrobu velkokapacitních matric s pevnou fází, jakou je například technologie mikropolí, zlepšila diagnostika potravinové alergie. V současnosti je možné analyzovat přecitlivělost na různé zdroje alergenů zprostředkovanou specifickými IgE stanovením specifických IgE proti alergenním proteinům či glykoproteinům. Tato změna nevedla jen ke zpřesnění diagnostiky senzibilizace, ale umožnila nám také vysvětlit různá rizika senzibilizace na určité molekuly, v mnoha případech fenomén zkřížené reaktivity, a dokonce může korigovat námi poskytovaná klinická doporučení. Pro přesnější vyhodnocení možností této nové techniky však bude třeba uskutečnit další studie.

The determination of specific IgE (sIgE) against allergenic components fixed in a solid support that is provided as a microarray of high capacity and allows a more precise evaluation in the food allergy diagnosis. In this review, we will analyze the results obtained to date with this technology applied to the component-based diagnosis of food allergy. RECENT FINDINGS: Microarrays of proteins or glycoproteins allow us to know the profile of sensitization of a patient with food allergy. At present, a commercially available technique exists which allows sIgE to be detected against 103 allergenic molecules. Several laboratories worldwide have explored and optimized this technique for few allergen extracts and the results have been promising with high reliabilities and sensitivities and above all, good correlations with previous existing conventional assays. SUMMARY: In recent years, as a result of advances in molecular biology, together with the development of new technologies of producing high-capacity solid-phase matrices such as microarrays, the diagnosis of food allergy has improved and the basic situation of analyzing sIgE against an allergenic source has now become real the possibility of analyzing sIgE against an allergenic protein or glycoprotein. This change has not only led to a more precise diagnosis of sensitization, but can also be used to explain the different hazards of certain molecular sensitizations, crossreactivity phenomena in many cases and can even change the clinical management according to the information provided. Further studies are clearly needed to evaluate more precisely the scope of this new technique.

• MeSH
• Allergens immunology   MeSH
• Financing, Organized MeSH
• Immunization MeSH
• Immunoglobulin E blood   MeSH
• Immunosorbent Techniques MeSH
• Humans MeSH
• Microarray Analysis MeSH
• Food Hypersensitivity diagnosis   immunology   MeSH
• Reproducibility of Results MeSH
• Sensitivity and Specificity MeSH
• Cross Reactions MeSH
• Check Tag
• Humans MeSH
• Publication type
• Review MeSH

• MeSH
• DNA, Neoplasm MeSH
• Genetic Techniques MeSH
• Clinical Laboratory Techniques MeSH
• Tumor Suppressor Protein p53 MeSH

cDNA microarray technology is widely used in various biological and medical disciplines to determine gene expression profiles. Unfortunately, this technology requires a large quantity of input RNA. Since there is an increasing need for more precise analyses of defined cell subpopulations with low cell counts, working protocols using a minimal number of input cells are required. Optimal RNA isolation and its accurate amplification are crucial to the success of these protocols. The HL-60 cell line was used in the search for a suitable protocol that can be used for clinical samples of CD34+ haematopoietic cells obtained from bone marrow. The goal was to discover the best method for isolating and amplifying RNA from a small number of cells. Our evaluation of various methods and kits available in the market revealed that the combination of RNAqueous™ Kit for RNA isolation and the SenseAmp Plus Kit for one-round RNA amplification produced the best results. This article presents a verified protocol describing a reliable and reproducible method for obtaining enough input RNA for microarray experiments when the number of cells is limited.

• MeSH
• Financing, Organized MeSH
• HL-60 Cells MeSH
• Cells, Cultured MeSH
• Humans MeSH
• RNA genetics   isolation & purification   MeSH
• Oligonucleotide Array Sequence Analysis methods   MeSH
• Nucleic Acid Amplification Techniques methods   MeSH
• Check Tag
• Humans MeSH

• MeSH
• Cells MeSH
• Microscopy, Fluorescence MeSH
• Microarray Analysis MeSH
• Image Processing, Computer-Assisted MeSH
• Tissues MeSH

Background: Microarray technologies are used to measure the simultaneous expression of a certain set of thousands of genes based on ribonucleic acid (RNA) obtained from a biological sample. We are interested in several statistical analyses such as 1) finding differentially expressed genes between or among several experimental groups, 2) finding a small number of genes allowing for the correct classification of a sample in a certain group, and 3) finding relations among genes. Objectives: Gene expression data are high dimensional, and this fact complicates their analysis because we are able to perform only a few samples (e.g. the peripheral blood from a limited number of patients) for a certain set of thousands of genes. The main purpose of this paper is to present the shrinkage estimator and show its application in different statistical analyses. Methods: The shrinkage approach relates to the shift of a certain value of a classic estimator towards a certain value of a specified target estimator. More precisely, the shrinkage estimator is the weighted average of the classic estimator and the target estimator. Results: The benefit of the shrinkage estimator is that it improves the mean squared error (MSE) as compared to a classic estimator. The MSE combines the measure of an estimator’s bias away from its true unknown value and the measure of the estimator’s variability. The shrinkage estimator is a biased estimator but has a lower variability. Conclusions: The shrinkage estimator can be considered as a promising estimator for analyzing high dimensional gene expression data.

• MeSH
• Gene Expression * genetics   MeSH
• Humans MeSH
• Microarray Analysis * methods   statistics & numerical data   MeSH
• RNA * genetics   MeSH
• Models, Statistical MeSH
• Check Tag
• Humans MeSH
• Publication type
• Research Support, Non-U.S. Gov't MeSH