Multiple Gene Expression Analysis Research Articles

Gene Expression profile of Staphylococcal Enterotoxin B and Lipopolysaccharide induced human PBMCs

Staphylococcal enterotoxin B (SEB), an exotoxin produced by staphylococcal aureus, is commonly associated with food poisoning and capable of triggering flu‐like symptoms resembling those of endotoxins – such as lipopolysaccharide (LPS) released from Gram‐negative bacterial cell walls. Eventhough both exhibit similar symptoms, each has its unique gene expression profile. Here we attempt to distinguish two toxins that display similar symptoms using multiple gene expression analysis techniques in a time dependent manner. Thirty genes that showed opposite expression patterns when exposed to the two toxins using micro‐array analysis were further analysed using multiple gene quantitation techniques such as RT‐PCR and real time PCR. These expression patterns were further validated by investigating the protein expression pattern. We believe that this study will not only allow us to confirm the results obtained through micro‐array experiments but also to better predict, identify and isolate varies exposures in a rapid and an accurate manner.

Integrated Analysis of Multiple Gene Expression Profiling Datasets Revealed Novel Gene Signatures and Molecular Markers in Nasopharyngeal Carcinoma

To identify the novel gene signatures and molecular markers of nasopharyngeal carcinoma (NPC) by integrated bioinformatics analysis of multiple gene expression profiling datasets. Seven published gene expression profiling studies and one of our unpublished works were reanalyzed to identify the common significantly dysregulated (CSD) genes in NPC. Overrepresentation analysis of cytogenetic bands, Gene Ontology (GO) categories, pathways were used to explore CSD genes functionally associated with carcinogenesis. The protein expressions of selected CSD genes were examined by immunohistochemistry on tissue microarrays, and the correlations of their expressions with clinical outcomes were evaluated. Using the criteria (genes reported deregulated in more than one study), a total of 962 genes were identified as the CSD genes in NPC. Four upregulated (BUB1B, CCND2, CENPF, and MAD2L1) and two downregulated (LTF and SLPI) genes were markedly reported in six studies. The enrichments of chromosome aberrations were 2q23, 2q31, 7p15, 12q15, 12q22, 18q11, and 18q12 in upregulated genes and 14q32 and 16q13 in downregulated genes. The activated GO categories and pathways related to proliferation, adhesion, invasion, and downregulated immune response had been functionally associated with NPC. SLPI significantly downregulated in nasopharyngeal adenocarcinoma. Furthermore, the high expression of BUB1B or CENPF was associated with poor overall survival of patients. It was first clearly identified the dysregulated expression of BUB1B and SLPI in NPC tissues. Further studies of the CSD genes as gene signatures and molecular markers of NPC might improve the understanding of the disease and identify new therapeutic targets.

Postnatal maternal separation modifies the response to an obesogenic diet in adulthood

SUMMARYAn early-life adverse environment has been implicated in the susceptibility to different diseases in adulthood, such as mental disorders, diabetes and obesity. We analyzed the effects of a high-fat sucrose (HFS) diet for 35 days in adult female rats that had experienced 180 minutes daily of maternal separation (MS) during lactancy. Changes in the obesity phenotype, biochemical profile, levels of glucocorticoid metabolism biomarkers, and the expression of different obesity- and glucocorticoid-metabolism-related genes were analyzed in periovaric adipose tissue. HFS intake increased body weight, adiposity and serum leptin levels, whereas MS decreased fat pad masses but only in rats fed an HFS diet. MS reduced insulin resistance markers but only in chow-fed rats. Corticosterone and estradiol serum levels did not change in this experimental model. A multiple gene expression analysis revealed that the expression of adiponutrin (Adpn) was increased owing to MS, and an interaction between HFS diet intake and MS was observed in the mRNA levels of leptin (Lep) and peroxisome proliferator-activated receptor gamma coactivator 1 alpha (Ppargc1a). These results revealed that early-life stress affects the response to an HFS diet later in life, and that this response can lead to phenotype and transcriptomic changes.

New gene targets for glucagon-like peptide-1 during embryonic development and in undifferentiated pluripotent cells

In humans, glucagon-like peptide (GLP-1) functions during adult life as an incretin hormone with anorexigenic and antidiabetogenic properties. Also, the therapeutic potential of GLP-1 in preventing the adipocyte hyperplasia associated with obesity and in bolstering the maintenance of human mesenchymal stem cell (hMSC) stores by promoting the proliferation and cytoprotection of hMSC seems to be relevant. Since these observations suggest a role for GLP-1 during developmental processes, the aim of the present work was to characterize GLP-1 in early development as well as its gene targets in mouse embryonic stem (mES) cells. Mouse embryos E6, E8, and E10.5 and pluripotent mES were used for the inmunodetection of GLP-1 and GLP-1 receptor. Quantitative real-time PCR was used to determine the expression levels of GLP-1R in several tissues from E12.5 mouse embryos. Additionally, GLP-1 gene targets were studied in mES by multiple gene expression analyses. GLP-1 and its receptors were identified in mES and during embryonic development. In pluripotent mES, GLP-1 modified the expression of endodermal, ectodermal, and mesodermal gene markers as well as sonic hedgehog, noggin, members of the fibroblast and hepatic growth factor families, and others involved in pancreatic development. Additionally, GLP-1 promoted the expression of the antiapoptotic gene bcl2 and at the same time reduced proapoptotic caspase genes. Our results indicate that apart from the effects and therapeutic benefits of GLP-1 in adulthood, it may have additional gene targets in mES cells during embryonic life. Furthermore, the pathophysiological implications of GLP-1 imbalance in adulthood may have a counterpart during development.

Immune profiling by multiple gene expression analysis in patients at-risk and with type 1 diabetes.

There is a need for biomarkers to monitor the development and progression of type 1 DM. We analyzed mRNA expression levels for granzyme B, perforin, fas ligand, TNF-α, IFN-γ, Foxp3, IL-10, TGF-β, IL-4, IL-6, IL-17, Activation-induced cytidine deaminase (AID) and Immunoglobulin G gamma chain (IgG<gamma>) genes in peripheral blood of at-risk, new-onset and long-term type 1 DM , and healthy controls. The majority of the genes were suppressed in long-term type 1 DM compared to controls and new-onset patients. IFN-γ, IL-4 and IL-10 mRNA levels were significantly higher in new-onset compared to at-risk and long-term groups. There was decreased mRNA expression for AID and IgG<gamma> and up-regulation of IFN-γ with age in controls. Data suggest an overall depressed immunity in long-term type 1 DM. Increased gene expression levels for IFN-γ, IL-4 and IL-10 in new-onset patients from at-risk patients might be used as potential markers for progression of the disease.

Multiple Gene Expression Analyses in Human Lymphoid Tissues by TaqMan Low-density Array Using Amplified RNA Isolated From Paraffin-Embedded Samples

In this study, we examined whether small amounts of RNA available from fixed and paraffin-embedded samples could be increased and quantified while conserving the gene expression profile. To this aim, RNA was extracted by a modified version of an earlier described method and was amplified with a linear amplification system. Furthermore, using TaqMan low-density arrays technology, the expression of multiple genes was correlated in matched snap-frozen and paraffin-embedded tissues. The entire technical procedure was assessed on 7 reactive lymph nodes. The results of the study showed that highly degraded RNA could be amplified to a mean of a 1200 to 3500-fold increase. Furthermore, the amount of cDNA and the polymerase chain reaction amplification efficiency was higher in amplified RNA than in unamplified RNA (89% vs. 82%). The analysis of Ct and DeltaCt values showed strong correlations in matched amplified versus unamplified, fixed and frozen samples, thus demonstrating the conservation of gene expression in amplified RNA. We conclude that small amounts of RNA from paraffin-embedded tissues can be successfully amplified without altering the gene expression. Consequently, TaqMan low-density array technology could be used successfully for the analysis of archival fixed and paraffin-embedded lymphoid tissue.

Evaluation of a low density DNA microarray for small B-cell non-Hodgkin lymphoma differential diagnosis

Lymphomas are classified according to the World Health Organisation (WHO) classification which defines subtypes on the basis of clinical, morphological, immunophenotypic, molecular and cytogenetic criteria. Differential diagnosis of the subtypes is sometimes difficult, especially for small B-cell lymphoma (SBCL). Standardisation of molecular genetic assays using multiple gene expression analysis by microarrays could be a useful complement to the current diagnosis. The aim of the present study was to develop a low density DNA microarray for the analysis of 107 genes associated with B-cell non-Hodgkin lymphoma and to evaluate its performance in the diagnosis of SBCL. A predictive tool based on Fisher discriminant analysis using a training set of 40 patients including four different subtypes (follicular lymphoma n = 15, mantle cell lymphoma n = 7, B-cell chronic lymphocytic leukemia n = 6 and splenic marginal zone lymphoma n = 12) was designed. A short additional preliminary analysis to gauge the accuracy of this signature was then performed on an external set of nine patients. Using this model, eight of nine of those samples were classified successfully. This pilot study demonstrates that such a microarray tool may be a promising diagnostic approach for small B-cell non-Hodgkin lymphoma.

Leprosy pathogenetic background: a review and lessons from other mycobacterial diseases

Leprosy is a disease caused by Mycobacterium leprae that initially affects the peripheral nervous system with patients exhibiting contrasting clinical, immunological, and pathological manifestations despite minimal genetic variation among bacilli isolates. Its clinical manifestations are related to M. leprae survival, innate and acquired immune responses, and interactions between host and bacterial proteins, preventing their invasion and infection, or promoting their development and pathogenesis. The complex molecular interactions in affected individuals influenced by the pathogenetic background will be explored in this review. However, the great genetic diversity imposes difficulty for understanding disease development, and it is likely that many factors and metabolic pathways regulating the immense and contrasting symptomatology will yet be revealed. Four pathways may play a central role in leprosy, including the TLR/LIR-7, VDR, TNF-alpha, and TGF-beta1 for which a large amount of gene polymorphisms have been described that could potentially affect the clinical outcome. Cross-talk pathways may significantly change the course of the disease, depending on the specific disequilibrium of genic homeostasis, which is highly dependent on the environment, antigens that are presented to the host cell, and specific polymorphisms that interact with other genes, external factors, and pathogen survival, culminating in leprosy occurrence. Currently, the microarray-based genomic survey of gene polymorphisms, multiple gene expression analyses, and proteomic technologies, such as mass spectrometry and phage display applied in the discovery of antigens, represent a great potential for evaluating individual responses of leprosy patients and contacts to predict the outcome and progression of the disease. At present, none of the genes is good prognostic marker; however, in the near future we may use multiple targets to predict infection and leprosy development.

A murine model for human immune thrombocytopenic purpura and comparative analysis of multiple gene expression in bone marrow and spleen

Homeostasis of platelet number in human and other mammals is well maintained for prevention of minor bleeding and for other immunological functions, but the exact molecular mechanism responsible for immune thrombocytopenic purpura (ITP) has not been fully understood. In an effort to identify genetic factors involved in initiation of platelet production in response to bleeding injury or platelet destruction, we have successfully generated an animal model of human ITP via intraperitoneal injection of anti-platelet antibody into the Balb/c mouse. Platelet counts were dropped dramatically in animals that received antibody injection within 4 h, maintained at the minimum level for a period of 44 h, started to rebound after 48 h, and reached to the maximum at 144 h (6 days). Final homeostasis reached at approximately 408 h (17 days), following a minor cycle of platelet number fluctuation. Using semi-quantitative RT-PCR, we assessed and compared mRNA level of CD41, c-myb, c-mpl, caspase-3, caspase-9, GATA-1, and Bcl-xl in bone marrow and spleen. Alteration of mRNA expression was correlated with the change of platelet level, and an inverse relationship was found for expression of the genes between bone marrow and spleen. No transcription was detectable for any of the seven genes in bone marrow at the time when platelet number reached the maximum (144 h). In contrast, mRNA transcripts of the seven genes were found to be at the highest level in spleen tissue. This is the first study of simultaneous detection of multiple platelet related genes in a highly reproducible ITP animal model. Our results provided the supportive evidence that expression of the above seven genes are more related to negative regulation of platelet number in spleen tissue, at least in the model animals.

Time‐ and concentration‐dependent metabolic and genomic responses to exposure to resin acids in brown trout (Salmo trutta m. lacustris)

The presence of metabolically conjugated resin acids (RAs) in the bile is considered to be a sensitive indicator for exposure of fish to pulp and paper industry effluents; however, to our knowledge, no comprehensive kinetic study of this response has been made. Juvenile brown trout (Salmo trutta m. lacustris) were exposed to a waterborne mixture of seven RAs (wood rosin) in time (0.1-192.0 h; average concentration, 8 microg/L) and dose (average concentrations, 0, 0.6, 4, 14, and 78 microg/L; 10 d) series, and total RAs were analyzed in bile. In time-dependent exposure, total RAs in bile increased up to 24 h. In concentration-dependent exposure, RAs increased along with the concentration of RAs in water, revealing a high-capacity biotransformation and elimination system in trout liver. In concentration-dependent exposures, the effects on the hepatic transcriptome was studied using a high-density cDNA microarray, and dose-dependent changes were found in a large number of genes. Resin acids interfered with iron metabolism, as evidenced by the decrease in transcripts for iron transporters and heme-containing proteins. Expression of genes encoding for enzymes degrading reactive oxygen species also decreased. Coordinated down-regulation of the protein biosynthesis machinery could result from inhibition of the energy metabolism. A number of changes in gene expression indicated recovery and remodeling of hepatic tissues. We conclude that analysis of total RAs in the bile provides a sensitive and quantitative tool for assessing the exposure of fish to waterborne RAs, whereas multiple gene expression analyses are able to elucidate simultaneous cellular functions for use as potential biomarkers of RAs.

Adequate design of customized cDNA macroarray for convenient multiple gene expression analysis

To establish a convenient, cost-effective, and reasonably reliable method for monitoring multiple gene expression using customized membrane-based macroarray, we constructed a cDNA macroarray with multiple probes for 13 human vascular endothelial genes and assessed the accuracy of the macroarray measurements. For each gene, two cDNA probes (450-550 bp) were designed from different regions (coding region and 3'-untranslated region [3'-UTR], respectively) on the basis of simple criteria concerning length and sequence specificity and spotted on the macroarray. In addition, unmodified oligonucleotide probes (80 mer) targeted to a unique sequence from the coding region of each gene were spotted on the same macroarray. Using this macroarray, shear stress-induced mRNA expression changes were analyzed in human coronary artery endothelial cells. Comparison of the expression ratios obtained with those measured using quantitative real-time polymerase chain reaction (PCR) as a reference method revealed that cDNA probes designed from a sequence within the coding region provided a highly accurate expression profile, whereas results obtained from oligonucleotide probes showed no correlation with real-time PCR data, which might be caused by inadequate immobilization of oligonucletotide probes on the nylon membrane. In addition, we observed that cDNA probes targeting different regions of a gene yielded different signal intensities. Most cDNA probes designed from a sequence within the coding region showed detectable signals, whereas few cDNA probes designed from 3'-UTR did.

Multiple tissue gene expression analyses in Japanese medaka ( Oryzias latipes) exposed to hypoxia

Due in part to human population growth watersheds and coastal estuaries have been receiving increasing run-off of nutrients and genotoxins. As a consequence, the occurrences of nutrient-driven hypoxia in coastal waters appear to be increasing. Thus, understanding the molecular genetic response to hypoxia by model aquatic organisms is of interest both from environmental and physiological viewpoints. The major objectives of this study are to determine genome-wide gene expression profiles and to better understand how hypoxia influences global gene expression in medaka ( Oryzias latipes), a well utilized aquatic model species. Herein we detail our development of a microarray containing 8046 medaka unigenes and describe our experimental results for measuring gene expression changes in the brain, gill, and liver of hypoxia exposed fish. Using conservative selection criteria, we determined that 501 genes in the brain, 442 in the gill, and 715 in the liver were differentially expressed in medaka exposed to hypoxia. These differentially expressed genes fell into a number of biological gene ontology groups related to general metabolism, catabolism, RNA and protein metabolism, etc. Two biological pathways, ubiquitin-proteasome and phosphatidylinositol signaling, were significantly dysregulated in medaka upon hypoxia exposure. Comparative genomics between medaka and human identified several human orthologies associated with known diseases.

Multiple Gene Expression Analyses in Paraffin-Embedded Tissues by TaqMan Low-Density Array: Application to Hedgehog and Wnt Pathway Analysis in Ovarian Endometrioid Adenocarcinoma

Recent studies have shown the hedgehog and Wnt families of signaling proteins to be associated with tumor initiation, growth, and survival. However, these pathways remain unexplored in ovarian endometrioid adenocarcinoma (OEA). Here, we describe a novel TaqMan low-density array to examine the expression of 26 and 20 genes in the hedgehog and Wnt pathways, respectively, in six matched snap-frozen and formalin-fixed, paraffin-embedded (FPE) OEA specimens. Expression values were normalized to uninvolved ovarian epithelium. Gene expression in matched frozen and FPE tissues demonstrated significant concordance (r = 0.92, P < 0.0001). However, comparison of amplified and unamplified RNA from frozen OEA tissues revealed an altered molecular profile in amplified RNA. Amplification of RNA from FPE tissues was not successful. The expression of Desert hedgehog (DHH), Indian hedgehog (IHH), Hedge-hog interacting protein (HHIP), Wnt10B, Wnt9B, and Wnt inhibitory factor (WIF1) were tumor-specific with no detectable expression in normal ovarian epithelium. In addition, several genes were significantly (P < 0.025) down-regulated in OEA, including cyclin E2, Porcupine, c-Myc, and Axin 2 (4.8-, 3.6-, 2.9-, and 1.9-fold, respectively). TaqMan low-density array provides an effective multivariate technique for examining gene expression in RNA isolated from either snap-frozen or archival FPE tissues and can identify tumor-specific genes, possibly leading to novel treatments.

Tissue microarray analysis of multiple gene expression in intestinal metaplasia, dysplasia and carcinoma of the stomach

To study multiple gene expression patterns and their roles in the process of gastric carcinogenesis. Using a high-throughput tissue microarray technique, 169 specimens from gastric carcinomas, precursor lesions and normal mucosa were immunostained on a series of tissue chips for p53, p21(WAF1/CIP1) cyclin E, Bcl-2, c-met and mucin 5AC expression. The overexpression of p53 was observed in 10.7% of low-grade dysplasia (LGD), 38.1% of high-grade dysplasia (HGD) and 39.6% of intestinal type gastric carcinoma (IGC). Expression of p21(WAF1/CIP1) was found in 47.6% of incomplete intestinal metaplasia (IM), 36.7% of dysplasia (Dys) and 29.5% of IGC. The overexpression of cyclin E was more frequently present in carcinomas than in Dys (P < 0.05); moreover, high-level expression (> 25% in extent) of cyclin E was observed only among IGC. Abnormal Bcl-2 expression was present in 81.0% of incomplete IM, 69.4% of Dys and 22.9% of IGC. Along with progression of the lesion, the expression of c-met increased; in contrast, mucin 5AC decreased gradually. The specific expression pattern in incomplete IM was mucin 5AC+/Bcl-2+/p53-/cyclin E-, while mucin 5AC-/cyclin E+ was specific for IGC. p53 was useful for distinguishing LGD from HGD. High-level expression of cyclin E might be an indicator for malignant transformation of dysplasia.

Quantification of multiple gene expression in individual cells.

Quantitative gene expression analysis aims to define the gene expression patterns determining cell behavior. So far, these assessments can only be performed at the population level. Therefore, they determine the average gene expression within a population, overlooking possible cell-to-cell heterogeneity that could lead to different cell behaviors/cell fates. Understanding individual cell behavior requires multiple gene expression analyses of single cells, and may be fundamental for the understanding of all types of biological events and/or differentiation processes. We here describe a new reverse transcription-polymerase chain reaction (RT-PCR) approach allowing the simultaneous quantification of the expression of 20 genes in the same single cell. This method has broad application, in different species and any type of gene combination. RT efficiency is evaluated. Uniform and maximized amplification conditions for all genes are provided. Abundance relationships are maintained, allowing the precise quantification of the absolute number of mRNA molecules per cell, ranging from 2 to 1.28 x 10(9) for each individual gene. We evaluated the impact of this approach on functional genetic read-outs by studying an apparently homogeneous population (monoclonal T cells recovered 4 d after antigen stimulation), using either this method or conventional real-time RT-PCR. Single-cell studies revealed considerable cell-to-cell variation: All T cells did not express all individual genes. Gene coexpression patterns were very heterogeneous. mRNA copy numbers varied between different transcripts and in different cells. As a consequence, this single-cell assay introduces new and fundamental information regarding functional genomic read-outs. By comparison, we also show that conventional quantitative assays determining population averages supply insufficient information, and may even be highly misleading.

Integrated assessment by multiple gene expression analysis of quercetin bioactivity on anticancer-related mechanisms in colon cancer cells in vitro.

Many different mechanisms are involved in nutrient-related prevention of colon cancer. In this study, a comprehensive assessment of the spectrum of possible biological actions of the bioactive compound quercetin is made using multiple gene expression analysis. Quercetin is a flavonoid that can inhibit proliferation of tumor cells and reduce the number of aberrant crypt foci, although increase of number of colon tumors was also reported. In order to elucidate possible mechanisms involved in its mode of action the effect of quercetin on expression of 4000 human genes in Caco-2 cells was studied and related to functional effects. Caco-2 cells were exposed to 5 or 50 microM quercetin for 48 hours, differential expression of 4000 human genes was studied using microarrays and related to functional effects. Differentially expressed genes were categorized in seven functional groups: cell cycle and differentiation, apoptosis, tumor suppressor genes and oncogenes, cell adhesion and cell-cell interaction, transcription, signal transduction and energy metabolism. Also, cell proliferation and cell cycle distribution were measured. Quercetin (5 microM) downregulated expression of cell cycle genes (for example CDC6, CDK4 and cyclin D1), downregulated cell proliferation and induced cell cycle arrest in Caco-2 cells. After exposure to 50 microM quercetin cell proliferation decreased to 51.3% of control, and further decrease of the percentage of cells in the G1 phase coincided with an increase of the percentage of cells in the sub-G1 phase. Quercetin upregulated expression of several tumor suppressor genes. In addition, genes involved in signal transduction pathways like beta catenin/TCF signalling and MAPK signal transduction were influenced by quercetin. This study shows that large-scale gene expression analysis in combination with functional assays yields a considerable amount of information on (anti-)carcinogenic potential of food components like quercetin.

Gene expression changes in single dentate granule neurons after adrenalectomy of rats

Removal of corticosterone by adrenalectomy induces apoptosis 3 days later, in some, but not all, rat dentate granule cells. We hypothesized that individual dentate cells trigger specific gene expression profiles that partly determine their apoptosis susceptibility. RNA was collected from physiologically characterized granule cells at 2 or 3 days after adrenalectomy or sham operation, and linearly amplified. The amplified RNA was hybridized to cDNA clones of: (1) candidate genes earlier identified after adrenalectomy in whole hippocampi with SAGE; and (2) genes encoding growth factors and their receptors. We observed that based on the entire expression profile, cells relatively resistant to apoptosis 3 days after adrenalectomy clustered together with one-third of cells 2 days after adrenalectomy. Within the group of ADX cells, a limited number of transcript ratios were found to correlate—positively or negatively—with a known risk factor for apoptosis, calcium influx. The overall analysis of physiological properties and multiple gene expression in single cells can narrow down the number of critical genes involved in apoptosis identified with large scale gene screening methods and allows a first impression of their role as being a potential risk factor or neuroprotective.

Human cDNA expression arrays in normal and malignant monocytes

New technologies have recently become available with complementary DNA (cDNA) microarrays, which allow simultaneous analysis of multiple cell gene expression. In hematological malignancies few data have been reported so far. We decided to investigate the usefulness of this challenging methodology in combination with immunomagnetic cell separation techniques. For that purpose, we performed a series of consecutive microarrays on different cell populations. Our samples consisted of monocytes from healthy donors, AMML and CMML patients. As reference we used cells from the KBM3 monocytic leukemia cell line. Cell separations based both on negative and positive selection were tested, in order to verify possibility of gene activation or suppression elicited by the interaction of monoclonal antibodies and surface antigens. After density gradient-based mononuclear cell enrichment, CD14+ cells were sorted using the MACS recommended procedures. FACS analysis was performed on separation products to test the purity of monocytic populations. Both mRNA and total RNA were tested for some of the samples, 32P-radiolabeled cDNA was synthesized and hybridized to Atlas Human cDNA expression array (Clontech) encompassing 588 selected genes. We were able to identify some major recurrent differences between normal and CMML monocytes. Results obtained with mRNA and total RNA were comparable. Negatively-isolated CMML monocytes showed an overexpression of chemokine genes such as migration inhibitory factor-related proteins 8 (MRP8) and 14 (MRP14) and a downregulation of the interleukin-13 (IL-13) precursor gene. Interestingly, low level of expression of some of these genes appears to be upregulated by positive selection performed by CD14-conjugated microbeads in normal monocytes, while no changes were observed in negatively selected monocytes. While human cDNA expression arrays seem to represent a reliable tool for the study of gene expression profiles, we experience some variability between different patients. In conclusion, our results demonstrate that cell separation methodology has an impact in the expression of certain genes and must be taken into consideration. Besides a statistical evaluation of the results, strict controls are always recommended.

Multiple gene expression analysis reveals distinct differences between G2 and G3 stage breast cancers, and correlations of PKC eta with MDR1, MRP and LRP gene expression.

A possible link between protein kinase C (PKC) and P-glycoprotein (P-gp)-mediated-multidrug resistance (MDR) was assumed from studies on MDR cell lines selected in vitro. The functional relevance of PKC for the MDR phenotype remains unclear, and the involvement of a particular PKC isozyme in clinically occurring drug resistance is not known. Recently, we have demonstrated significant correlations between the expression levels of the PKC eta isozyme and the MDR1 or MRP (multidrug resistance-associated protein) genes in blasts from patients with acute myelogenous leukaemia (AML) and in ascites cell aspirates from ovarian cancer patients. To extend these findings to further types of human tumours we analysed specimens from 64 patients with primary breast cancer for their individual expression levels of several MDR-associated genes (MDR1, MRP, LRP (lung cancer resistance-related protein), topoisomerase (Topo) II alpha/IIbeta, cyclin A and the PKC isozyme genes (alpha, beta1, beta2, eta, theta, and mu) by a cDNA-PCR approach. We found significantly enhanced mean values for MRP, LRP and PKC eta gene expression, but significantly decreased Topo II alpha and cyclin A gene expression levels in G2 tumours compared with G3. Remarkably, significant positive correlations between the MDR1, MRP or LRP gene expression levels and PKC eta were determined: MDR1/PKC eta (rs = +0.6451, P < 0.0001) n = 62; MRP/PKC eta (rs = +0.5454, P < 0.0001) n = 63; LRP/PKC eta (rs = +0.5436, P < 0.0001) n = 62; MRP/LRP (rs = +0.7703, P < 0.0001) and n = 62, MDR1/MRP (rs = +0.5042, P < 0.0001) n = 62. Our findings point to the occurrence of a multifactorial MDR in the clinics and to PKC eta as a possible key regulatory factor for up-regulation of a series of MDR-associated genes in different types of tumours.

Isolation of single immunohistochemically identified whole neuronal cell bodies from post-mortem human brain for simultaneous analysis of multiple gene expression

In Alzheimer's disease (AD), one cell in the brain may clearly be affected, while an adjacent cell appears healthy or unaffected. Previous technology has allowed us to examine one message at a time, at the level of a single cell (in situ hybridization, ISH), or multiple messages in a heterogeneous population of cells (Northern analysis). We have developed a methodology to build up a profile of multiple mRNA expression in single, whole, post-mortem cells that have been immunohistochemically (IHC) characterized. Fresh post-mortem tissue is spread into a layer one cell thick and fixed. Neurons are identified using an antibody to neurofilament and isolated using a micropipette. The mRNA is reverse transcribed and PCR carried out to confirm that material is present. A radioactively labeled antisense aRNA probe, which is representative of the messages contained in the cell is then amplified. This aRNA is used as a probe for a reverse Northern blot, allowing us to profile many genes from one cell at the same time. This technology has the potential to be applied to a wide variety of diseases encompassing many different cell types.