This study combines mRNA and protein analysis using cDNA and antibody microarray techniques, respectively. These create a novel, integrated perspective into cellular molecular profiles. The aims of this study were to establish a reliable way of integrating these two approaches in order to obtain complex molecular profiles of the cell and to find suitable methods to normalize the data obtained using these approaches.
Antibody microarray and cDNA microarray techniques were used to study expression alterations in HL-60 cells that were differentiated into granulocytes using all-trans retinoic acid (ATRA). We selected this model to evaluate this combined profiling technique because the expression levels of most of the mRNA and protein species in these cells are not altered; therefore it is easier to track and define those species that are changed. The proteins whose levels were altered included c-myc, c-jun, Pyk2, FAK, PKC, TRF1, NF-kappaB and certain caspase types. These proteins are involved in apoptosis and hematopoietic differentiation pathways, and some have also been reported to have oncogenic potential. We compared the results obtained using the two methods, verified them by immunoblotting analysis, and devised normalization approaches.
This is one of the first demonstrations that a combination of antibody microarray and cDNA microarray techniques is required for complex molecular profiling of cells based on multiple parameters. This approach allows a more detailed molecular phenotype of the given sample to be obtained. The results obtained using a combination of the two profiling methods are consistent with those from previous studies that used more traditional methods.
Keywords: microarray, cell profiling, protein expression, mRNA expression, HL-60.- MeSH
- čipová analýza proteinů MeSH
- financování organizované MeSH
- fokální adhezní kinasa 2 analýza MeSH
- geny myc MeSH
- HL-60 buňky MeSH
- lidé MeSH
- messenger RNA analýza MeSH
- protein TRF1 analýza MeSH
- sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů MeSH
- tretinoin farmakologie MeSH
- Check Tag
- lidé MeSH
To understand how genes are distributed on chromosomes we bring new insights into gene positional clustering and its properties. We have made a large-scale analysis of three types of differentiation and we observed that genes that subsequently enter into different cell processes are positionally clustered on chromosomes. Genes from the clusters are transcribed subsequently with respect to time kinetics and also to position. This means that the genes related to a cellular process are clustered together, independent of the period of time during which they are active and important for the process. Our results also demonstrate not only that there are general regions of increased or decreased levels of gene expression, but also that, in fact, in some chromosome regions we can find clustering of genes related to specific cell processes. The results provided in this paper also support the theory of "transcription factories" and show that transcription of genes from the clusters is managed by softer epigenetic mechanisms.
- MeSH
- buněčná diferenciace genetika MeSH
- buňky K562 MeSH
- DNA primery genetika MeSH
- financování organizované MeSH
- genetická transkripce MeSH
- granulocyty cytologie metabolismus MeSH
- HL-60 buňky MeSH
- lidé MeSH
- megakaryocyty cytologie metabolismus MeSH
- monocyty cytologie metabolismus MeSH
- multigenová rodina MeSH
- myelopoéza genetika MeSH
- sekvence nukleotidů MeSH
- sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů MeSH
- stanovení celkové genové exprese MeSH
- trombopoéza genetika MeSH
- Check Tag
- lidé MeSH
V této publikaci přinášíme přehled vědeckých prací, ve kterých nám technologie cDNA microarrays umožnila nové náhledy do procesů maligní transformace. Díky této technologii dnes můžeme sledovat v jeden okamžik expresní aktivitu tisíců genů. S pomocí vhodných nástrojů můžeme naměřené hodnoty dávat do souvislostí, které byly ještě nedávno prakticky nemyslitelné. V naší laboratoři jsme vyvinuli nové nástroje a postupy pro analýzu microarrays a pro zpracování výsledků do podoby použitelné v onkologickém výzkumu. Pro sledování některých charakteristik je nutné použít vizualizační nástroje. Například pro sledování aktivity určitých oblastí chromosomů jsou velmi vhodné transkripční mapy (dále TM). Implementovali jsme vlastní software pro generování TM a díky kombinaci jeho výstupů s dosavadními znalostmi jsme formulovali závěry našich experimentů. Intuitivním rozšířením TM jsme dosáhli mnoha dalších vizualizací např. hustoty aktivních genů, kumulativní exprese oblastí atp. Tyto nástroje by měly v budoucnosti pomáhat klinikům při diagnostice zhoubných malignit a určování následné léčby.
In this paper we bring a scientific review of studies in which cDNA microarrays technology allowed us to create new insights into malignant transformation processes. We can monitor parallel expression activity of thousands of genes using this technology. Using appropriate tools we can evaluate biological correlations that were practically impossible few years ago. We have developed new tools and techniques transforming the data into form suitable for oncological research. Special visualizations are necessary for some types of studies. E.g. transcription maps (TMs) are a proper tool for studying activity of chromosomal regions. We have implemented our own software for generating TMs. On the basis of combination of its outputs and knowledge gained so far we have formulated our conclusions. Using intuitive TM extensions we have obtained many useful visualizations, e.g. density of active genes, cumulative expression etc. These tools are supposed to help clinicians while forming diagnoses of malignant diseases and while treatment planning.
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- biomedicínský výzkum metody trendy MeSH
- C-DNA diagnostické užití MeSH
- financování organizované MeSH
- lékařská onkologie metody trendy MeSH
- leukemie diagnóza genetika imunologie MeSH
- lidé MeSH
- mikročipové analytické postupy metody využití MeSH
- nádory tračníku diagnóza genetika imunologie MeSH
- stanovení celkové genové exprese metody využití MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- přehledy MeSH
OBJECTIVE: Hematopoietic stem cells (enriched in fraction of CD34+ cells) have the ability to regenerate hematopoiesis in all of its lineages, and this potential is clinically used in transplanting bone marrow or peripheral blood stem cells. Our objective was to assemble a suitable method for evaluating gene expression in enriched populations of hematopoietic stem cells. We compared biologic properties of cells cultured ex vivo obtained using two different ways of immunomagnetic separation (positive selection of CD34+ cells and negative selection of Lin- cells) by means of a cDNA microarray technique. METHODS: CD34+ and Lin- cells were enriched from peripheral blood stem cell (PBSCs) grafts of patients with non-Hodgkin's lymphoma. Isolated cells were in the presence of cytokine PBSCs, Flt-3 ligand, interleukin-3, interleukin-6, and granulocyte colony-stimulating factor. At days 0, 4, 6, 8, 10, 12, and 14 cells were harvested and analyzed by cDNA microarrays. Total cell expansion, CD34+, colony-forming unit for granulocyte-macrophage and megakaryocytes expansion, vitality, and phenotype of cells were also analyzed. RESULTS: cDNA microarray analysis of cultured hematopoietic cells proved equivalence of the two enrichment methods for PBSC samples and helped us characterize differentiating cells cultured ex vivo. CONCLUSION: Our methodologic approach is helpful in characterizing cultured hematopoietic cells cultured ex vivo, but it is also suitable for more general purposes. Equivalence of CD34+ and Lin- selection methods from PBSC samples proved by cDNA microarray may have an implication for graft manipulation in an experimental setting of hematopoietic transplantation. Total cell expansion and colony formation and phenotype from CD34+ selected and from Lin- samples were comparable.
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- antigeny CD34 imunologie MeSH
- financování organizované MeSH
- imunofenotypizace MeSH
- imunomagnetická separace MeSH
- komplementární DNA MeSH
- lidé MeSH
- lymfom genetika imunologie terapie MeSH
- sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů MeSH
- transplantace kmenových buněk MeSH
- Check Tag
- lidé MeSH
The progression of colorectal cancer involves accumulation of various genetic and epigenetic events that dramatically change gene expression. The aim of this study was to investigate a possible new approach to the diagnosis of colorectal carcinoma patients, based on their gene expression profiles. Human 19K cDNA microarrays were used to analyze the gene expression profiles of 18 colorectal carcinoma patients. Transcriptome maps (TMs) were analyzed to detect chromosomal regions that could serve as potential diagnostic markers for colon cancer. A comparison of TMs showed chromosome regions with conserved changes of gene expression typical of colorectal cancer in general, and also patient-specific variable regions. We identified 195 genes with significantly altered expression in colon cancer. Functional analysis of the regulated genes distinguished three main categories: biological processes, cellular components, and molecular functions. We found that different patients had chromosome regions characterized by very similar changes of gene expression, probably linked to the most fundamental events in carcinogenesis. On the other hand, variable chromosome regions can be patient-specific. The variable regions may provide further information on the individual pathogenesis and prognosis of the patient. Comparison of TMs is proposed as a tool to facilitate diagnosis and treatment planning for individual patients.
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- DNA nádorová genetika MeSH
- financování organizované MeSH
- genetická transkripce MeSH
- kolorektální nádory diagnóza genetika MeSH
- lidé středního věku MeSH
- lidé MeSH
- mapování chromozomů MeSH
- nádorové biomarkery genetika MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů metody MeSH
- stanovení celkové genové exprese MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- MeSH
- čipová analýza proteinů metody MeSH
- diagnostické techniky molekulární metody MeSH
- DNA analýza MeSH
- dospělí MeSH
- finanční podpora výzkumu jako téma MeSH
- kolorektální nádory diagnóza MeSH
- lidé MeSH
- lymfatické metastázy diagnóza MeSH
- regulace genové exprese u nádorů MeSH
- technologie lékařská MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- MeSH
- exprese genu * MeSH
- faktor Xa MeSH
- interpretace statistických dat MeSH
- kolorektální nádory * genetika MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- lidé MeSH
- metastázy nádorů MeSH
- mikročipová analýza přístrojové vybavení MeSH
- nádorové biomarkery MeSH
- nikotinamid-N-methyltransferasa MeSH
- počítačové zpracování obrazu MeSH
- sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů * MeSH
- software MeSH
- statistika jako téma MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
