DNA Microarray Assay Research Articles

Detection and identification of enterohemorrhagic Escherichia coli O157:H7 and Vibrio cholerae O139 using oligonucleotide microarray

BackgroundThe rapid and accurate detection and identification of the new subtype of the pathogens is crucial for diagnosis, treatment and control of the contagious disease outbreak. Here, in this study, an approach to detect and identify Escherichia coli O157:H7 and Vibrio cholerae O139 was established using oligonucleotide microarray. We coupled multiplex PCR with oligonucleotide microarray to construct an assay suitable for simultaneous identification of two subtypes of the pathogens.ResultsThe stx1, stx2 gene and uidA gene having the specific mutant spot were chosen as the targets for Escherichia coli O157:H7, and meanwhile the ctxA, tcpA, and LPSgt gene for Vibrio cholerae O139. The oligonucleotide microarray was composed of eight probes including negative control and positive control from 16S rDNA gene. The six primers were designed to amplify target fragments in two triplex PCR, and then hybridized with oligonucleotide microarray. An internal control would be to run a PCR reaction in parallel. Multiplex PCR did not produce any non-specific amplicons when 149 related species or genera of standard bacteria were tested (100% specificity). In addition, Escherichia coli O157:H7 and Escherichia coli O157:non-H7, Vibrio cholerae O139 and Vibrio cholerae O1 had been discriminated respectively. Using recombinant plasmid and target pathogens, we were able to detect positive hybridization signals with 102 copies/μL and 103 cfu/mL per reaction.ConclusionThe DNA microarray assay reported here could detect and identify Escherichia coli O157:H7 and Vibrio cholerae O139, and furthermore the subtype was distinguished. This assay was a specific and sensitive tool for simultaneous detection and identification of the new subtype of two pathogens causing diarrhea in human.

Use of a DNA Microarray for Simultaneous Detection of Antibiotic Resistance Genes among Staphylococcal Clinical Isolates

We developed a multiplex asymmetric PCR (MAPCR)-based DNA microarray assay for characterization of the clinically relevant antibiotic resistance genes leading to penicillin, methicillin, aminoglycoside, macrolide, lincosamide, and streptogramin B (MLS(B)) resistance in staphylococci. The DNA-based assay involves detection of specific conserved regions of the mecA, blaZ (methicillin and penicillin resistance), aac(6')-Ie-aph(2'') (aminoglycoside resistance), ermA and ermC genes (MLS(B) resistance), and the msrA gene (macrolide and streptogramin B resistance). The microarray uses a variable sequence region of the 16S rRNA gene to broadly differentiate between Staphylococcus aureus and other coagulase-negative staphylococci (CoNS). The performance of the microarray was validated with a total of 178 clinically important S. aureus and 237 CoNS isolates, with correlations of 100% for S. aureus to CoNS discrimination and more than 90% for antibiotic resistance between the genotypic analysis determined by the microarray and the phenotype determined by standard methods of species identification and susceptibility testing. The major discrepant results were 17 mecA-positive CoNS and 60 aac(6')-Ie-aph(2'')-positive CoNS isolates measured by microarray that were susceptible to the corresponding antibiotics based on disk diffusion assay. Overall, this microarray-based assay offers a simultaneous, fast (< or =5 h), and accurate identification of antibiotic resistance genes from a single colony, as well as species classification. Our extensive validation of the microarray suggests that it may be a useful tool to complement phenotypic susceptibility testing in clinical laboratories and to survey the spread of antibiotic resistance determinants in epidemiological studies.

Use of multicellular tumor spheroids to dissect endothelial cell–tumor cell interactions: A role for T-cadherin in tumor angiogenesis

This study addresses establishment of an “in vitro” melanoma angiogenesis model using multicellular tumor spheroids (MCTS) of differentiated (HBL) or undifferentiated (NA8) melanoma cell lines. DNA microarray assay and qRT-PCR indicated upregulation of pro-angiogenic factors IL-8, VEGF, Ephrin A1 and ANGPTL4 in NA8-MCTSs (vs. monolayers) whereas these were absent in MCTS and monolayer cultures of HBL. Upon co-culture with endothelial cell line HMEC-1 NA8-MCTS attract, whereas HBL-MCTS repulse, HMEC-1. Overexpression of T-cadherin in HMEC-1 leads to their increased invasion and network formation within NA8-MCTS. Given an appropriate angiogenic tumor microenvironment, T-cadherin upregulation on endothelial cells may potentiate intratumoral angiogenesis.

Direct identification of chlamydiae from clinical samples using a DNA microarray assay—A validation study

While DNA microarrays have become a widely accepted tool for mRNA expression monitoring, their use in rapid diagnosis of bacterial and viral pathogens is only emerging. So far, insufficient sensitivity and high costs have been the major limiting factors preventing more widespread use of microarray platforms in direct testing of clinical samples. In the present study, a total of 339 samples, among them 293 clinical specimens from animals and humans, were examined by the ArrayTube™ (AT) DNA microarray assay to detect chlamydial DNA and identify the species of Chlamydia and Chlamydophila involved. Samples included nasal and conjunctival swabs, formalin-fixed, paraffin-embedded and fresh organ tissue, milk, feces and cell culture. Notably, the AT test was shown to detect mixed infections in clinical samples. The calculated median sensitivity of 0.81 over the entire panel of clinical samples was comparable to conventional 16S PCR, but slightly lower than real-time PCR and other PCR assays. However, when a panel of long-time stored swab samples was excluded from the calculation, the sensitivity was clearly higher (0.87) and equivalent to that of real-time PCR. Altogether, the data demonstrate the suitability of this DNA microarray assay for routine diagnosis.

Interferon‐gamma‐dependent mechanisms of mycobacteria‐induced pulmonary immunopathology: the role of angiostasis and CXCR3‐targeted chemokines for granuloma necrosis

The mechanisms leading to granuloma caseation, a hallmark of tuberculosis (TB) in humans, are poorly understood. Lung histopathology of C57BL/6 (WT) mice 16 weeks after aerosol infection with Mycobacterium avium strain TMC724 is uniquely characterized by centrally necrotizing granulomas, strongly resembling human TB lesions. However, IFN-gamma-deficient (GKO) and IFN-gamma-receptor-deficient (GRKO) mice did not develop granuloma necrosis following M. avium infection. Comparison of differentially expressed genes in infected WT and GKO lungs by DNA microarray and RNase protection assays revealed that the angiostatic chemokines CXCL9-11 were significantly reduced in GKO mice. In contrast, angiogenic mediators such as angiopoietin and vascular endothelial growth factor, and angiogenic chemokines such as CXCL2, CCL3, and CCL4, remained unchanged or were expressed at higher levels than in infected WT mice, suggesting impaired neovascularization of the granuloma as a possible mechanism for caseation in WT mice. Granuloma vascularization was significantly decreased in central, but not peripheral, areas of granulomas of infected WT compared to GKO mice. In contrast to GRKO mice, WT mice showed signs of severe hypoxia in cells immediately surrounding the necrotic core of granulomas as measured immunohistochemically with a reagent detecting pimonidazole adducts. To test the hypothesis that CXCR3, the common receptor for the angiostatic chemokines CXCL9-11, is involved in granuloma caseation, histomorphology was assessed in M. avium-infected mice deficient for CXCR3 (CXCR3-KO). 16 weeks after infection, these mice developed caseating granulomas similar to WT mice. We conclude that IFN-gamma causes a dysbalance between angiostatic and angiogenic mediators and a concomitant reduction in granuloma vascularization, but that CXCR3-targeted chemokines are not sufficient to induce granuloma necrosis in a mouse model of mycobacteria-induced immunopathology.

Mre11 mediates gene regulation in yeast spore development

Mre11, together with Rad50 and Xrs2/NBS, plays pivotal roles in homologous recombination, repair of DNA double strand breaks (DSBs), activation of damage-induced checkpoint, and telomere maintenance. Here we demonstrate that the absence of Mre11 in yeast causes specific effects on regulation of a class of meiotic genes for spore development. Using DNA microarray assays to analyze yeast mutants defective for meiotic DSB formation, we revealed that the meiotic expression profile in the mre11Delta cells was generally unaffected when compared to the one in the wild-type strain, although the activation of about 90 meiotic genes were severely and specifically impaired in early meiosis. These defects were confirmed by northern and lacZ reporter gene assays. Interestingly, a substantial portion of the severely affected genes includes genes responsible for spore wall biogenesis, the defects of which may account for the fragile spore wall phenotype of the mre11Delta strain. The transcriptional deficiency was not observed in other DSB mutants such as rad50Delta, xrs2Delta, spo11Delta, and spo11Y135F, suggesting the transcriptional defect in mre11Delta is due to neither lack of meiotic DSB formation, nor disintegrity of Mre11-Rad50-Xrs2 complex. In addition, the deficiency of mre11Delta in gene activation was not alleviated by the deletion of RAD24. Therefore, it is unlikely that DNA damage checkpoint activation by mre11Delta caused transcriptional deficiency. We also found that a C-terminus DNA binding domain truncation mutant (mre11DeltaC49), which has meiosis-specific defects, exhibited transcriptional defects as observed in mre11Delta, whereas an N-terminal phosphoesterase mutant (mre11D16A) does not. Taken together, we propose that Mre11 is involved in the regulation of a specific class of genes during spore development through its C-terminus domain.

Effect of Akt inhibition on scatter factor-regulated gene expression in DU-145 human prostate cancer cells

The cytokine scatter factor (SF) (hepatocyte growth factor) transduces various biologic actions, including cell motility, invasion, angiogenesis and apoptosis inhibition. The latter is relevant to understanding the role of SF in promoting tumor cell survival in different contexts, for example, detachment from basement membrane, growth in metastatic sites and responses to chemo- and radiotherapy. Previously, we showed that SF protects cells against apoptosis owing to DNA damage, by a mechanism involving phosphoinositol-3-kinase/c-Akt signaling. Here, we used DNA microarray assays to identify c-Akt-regulated genes that might contribute to cell protection. DU-145 human prostate cancer cells were transfected+/-a dominant-negative mutant Akt, treated+/-SF and analysed for gene expression using Affymetrix arrays. These studies identified SF-regulated genes for which induction was c-Akt-dependent vs -independent. Selected microarray findings were confirmed by semiquantitative and quantitative reverse transcription-polymerase chain reaction. We tested the contribution of four SF-inducible/c-Akt-dependent genes (AMPD3, EPHB2, MX1 and WNT4) to protection against adriamycin (a DNA topoisomerase IIalpha inhibitor) using RNA interference. Knockdown of each gene except EPHB2 caused a small but significant reduction in the SF cell protection. The lack of effect of EPHB2 knockdown may be due to the fact that DU-145 cells contain a single-mutant EPHB2 allele. A combination of three small interfering RNAs blocked most of the protection by SF in both DU-145 and T47D cells. These findings identify novel c-Akt-regulated genes, some of which contribute to SF-mediated cytoprotection.

Gene Expression Profiling Reveals Reproducible Human Lung Adenocarcinoma Subtypes in Multiple Independent Patient Cohorts

Published reports suggest that DNA microarrays identify clinically meaningful subtypes of lung adenocarcinomas not recognizable by other routine tests. This report is an investigation of the reproducibility of the reported tumor subtypes. Three independent cohorts of patients with lung cancer were evaluated using a variety of DNA microarray assays. Using the integrative correlations method, a subset of genes was selected, the reliability of which was acceptable across the different DNA microarray platforms. Tumor subtypes were selected using consensus clustering and genes distinguishing subtypes were identified using the weighted difference statistic. Gene lists were compared across cohorts using centroids and gene set enrichment analysis. Cohorts of 31, 72, and 128 adenocarcinomas were generated for a total of 231 microarrays, each with 2,553 reliable genes. Three adenocarcinoma subtypes were identified in each cohort. These were named bronchioid, squamoid, and magnoid according to their respective correlations with gene expression patterns from histologically defined bronchioalveolar carcinoma, squamous cell carcinoma, and large-cell carcinoma. Tumor subtypes were distinguishable by many hundreds of genes, and lists generated in one cohort were predictive of tumor subtypes in the two other cohorts. Tumor subtypes correlated with clinically relevant covariates, including stage-specific survival and metastatic pattern. Most notably, bronchioid tumors were correlated with improved survival in early-stage disease, whereas squamoid tumors were associated with better survival in advanced disease. DNA microarray analysis of lung adenocarcinomas identified reproducible tumor subtypes which differ significantly in clinically important behaviors such as stage-specific survival.

Overexpression ofMLH‐1andpsoriasingenes in cutaneous angiofibromas from tuberous sclerosis complex patients

Tuberous sclerosis complex (TSC) is associated with mutations in two likely tumor-suppressor genes (TSC1 and TSC2) and characterized by the development of tumor-like growths (angiofibromas) in a variety of tissues and organs, particularly brain and skin. Employing a DNA-microarray assay, able to detect mRNA production from 1176 different basic genes, we analyzed the gene-expression levels in a cutaneous hamartoma sample from a TSC patient. Altered gene expressions detected by microarray technology were further checked by quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) in the same material and in cutaneous hamartoma samples obtained from five other TSC patients. The results obtained by the microarray technology in one hamartoma specimen, confirmed by the RT-PCR results obtained in the same material and in five other hamartoma specimens, demonstrated that TSC-related angiofibromas exhibit significant mRNA overexpression of two genes, represented by MLH-1 and psoriasin. The overexpression of MLH-1, which codes for a DNA mismatch repair protein, and psoriasin, which codes for a specific chemoattractant factor for CD4+ T cells, implicated in the pathogenesis of inflammatory skin disease, and expressed in excess during abnormal pathways of cell growth, may shed light on the pathogenesis of the proliferative skin lesion.

Identification of genes that modulate sensitivity of U373MG glioblastoma cells to cis-platinum

Scatter factor (hepatocyte growth factor) and its receptor c-Met are increasingly expressed during progression from low-grade to high-grade gliomas. Scatter factor/c-Met signaling induces glioma cell motility, invasion, angiogenesis and resistance to DNA-damaging agents. The latter is relevant to the understanding of the resistance of human gliomas to chemotherapy and radiotherapy. The goal of this study was to identify a set of genes that may contribute to scatter factor-mediated protection of U373MG cells against cis-platinum, a DNA cross-linking agent. We used DNA microarray assays, confirmatory semiquantitative reverse transcription-polymerase chain reaction analysis and functional assays to identify genes involved in the scatter factor-induced resistance of U373MG to cis-platinum. We identified a group of genes that are overexpressed in cells treated with scatter factor plus cis-platinum relative to cells treated with cis-platinum alone and confirmed some of these gene expression alterations by reverse transcription-polymerase chain reaction. Inhibiting the expression of three of these genes--polycystic kidney disease 1, amplified in breast cancer 1 and DEAD/H box helicase 21--using small interfering RNAs reduced survival of cis-platinum-treated cells and partially reversed the scatter factor protection against cis-platinum. Dominant-negative Akt and IkappaB super-repressor expression vectors inhibited the scatter factor protection, and abrogated the ability of scatter factor to alter the expression of DEAD/H box helicase 21 and polycystin (PKD1) within the context of cis-platinum exposure. The Akt and nuclear factor-kappaB inhibitors had no effect on amplified in breast cancer 1 expression. These studies implicate DEAD/H box helicase 21, polycystin (PKD1) and amplified in breast cancer 1 as novel transcription-dependent regulators of scatter factor-mediated glioma cell protection against cytotoxic death, and identify other potential regulators for future study.

Molecular Zipper: a fluorescent probe for real-time isothermal DNA amplification

Rolling-circle amplification (RCA) and ramification amplification (RAM, also known as hyperbranched RCA) are isothermal nucleic acid amplification technologies that have gained a great application in in situ signal amplification, DNA and protein microarray assays, single nucleotide polymorphism detection, as well as clinical diagnosis. Real-time detection of RCA or RAM products has been a challenge because of most real-time detection systems, including Taqman and Molecular Beacon, are designed for thermal cycling-based DNA amplification technology. In the present study, we describe a novel fluorescent probe construct, termed molecular zipper, which is specially designed for quantifying target DNA by real-time monitoring RAM reactions. Our results showed that the molecular zipper has very low background fluorescence due to the strong interaction between two strands. Once it is incorporated into the RAM products its double strand region is opened by displacement, therefore, its fluorophore releases a fluorescent signal. Applying the molecular zipper in RAM assay, we were able to detect as few as 10 molecules within 90 min reaction. A linear relationship was observed between initial input of targets and threshold time (R2 = 0.985). These results indicate that molecular zipper can be applied to real-time monitoring and qualification of RAM reaction, implying an amenable method for automatic RAM-based diagnostic assays.

Chemoresistance-Related Genes Profile in Specimens Obtained By Endoscopic Ultrasound-Guided Fine-Needle Aspiration (EUS-FNA)

Background: Chemotherapy using gemcitabine or 5-FU in advanced pancreatic cancer is often limited by development of side effects and drug resistance. The inter-individual variability of these events may have a genetic basis. DNA microarray systems can measure simultaneous expression of multiple genes in cancer patients. EUS-FNA, which is established as the preferred method to confirm a diagnosis of pancreatic cancer, can also be used to provide pancreatic cancer tissue for microarray analysis. Methods: Pancreatic cancer tissue was obtained from 10 patients with advanced disease by EUS-FNA, using a 22-gauge aspiration needle or 19-gauge trucut needle. Total mRNA was harvested from each sample which was amplified to provide obtain adequate mRNA for the analysis. The gene profile of DNA array was restricted to 133 genes including chemoresistance-related genes. (Focused DNA ArrayTM, Taiho Pharmaceutical Co., Ltd., Tokushima, Japan). Result: In all cases, adequate tissue samples were obtained and DNA microarray assays were successfully achieved. The gene amounts were expressed as the relative values to the individual house keeping gene amount. The mean ± SD of the 10 samples were as follows. Gemcitabine-resistance-related genes: dCMP deaminase, 4.72 ± 1.28; RRM1, 0.45 ± 0.10. 5-FU-resistance-related genes: TS, 7.50 ± 3.38; DPD, 1.41 ± 0.30. One of four patients treated with gemcitabine alone showed partial response (PR) and has survived for 8 months (M). The patient with the highest dCMP deaminase (7.41) among 10 patients and the other patient with the highest RRM1 (0.64) resulted in progress disease (PD) and died within 8 M. One of two patients treated with gemcitabine + S-1 (oral fluorouracil derivative containing a DPD inhibitor) showed PR and has been alive for 11 months. The dCMP deaminase, RRM1, and DPD were almost same and average level in the two patients, however, the TS level (8.07) in PD patient was higher compared to that (4.9) in PR patient. Conclusion: DNA microarray-based gene expression profiling combined with EUS-FNA may be a promising tool to predict chemoresistance in advanced pancreatic cancer. Such information may be useful to select individualizing chemotherapeutic regimens based on each patient's genotype.

Identification of gene networks modulated by activin in LßT2 cells using DNA microarray analysis

Activins, members of the TGFbeta family of proteins, are widely expressed in a variety of tissues. First identified based on their ability to regulate biosynthesis and secretion of follicle-stimulating hormone (FSH), activins have also been shown to modulate development, cell growth, apoptosis, and inflammation. Despite their many known functions, the precise mechanisms and downstream signaling pathways by which activins mediate their diverse effects remain unknown. We have used a DNA microarray assay to identify genes that are regulated by activin, alone or in combination with gonadotropin-releasing hormone (GnRH), another major regulator of FSH, in a murine gonadotrope-derived cell line (LbetaT2). We used mRNA from these cells to screen Affymetrix Mu74av2 mouse Gene Chip oligonucleotide microarrays, representing approximately 12,400 mouse genes. Treatment of LbetaT2 cells with activin A, a gonadotropin-releasing hormone agonist (GnRHA) or activin A plus GnRHA resulted in alterations in levels of gene expression that ranged in magnitude from 15 to 67-fold. Data analysis identified 268 transcripts that were up- or down-regulated by two-fold or more. Distinct sets of genes were affected by treatment with activin, GnRHA and activin plus GnRHA, suggesting interactions between activin and GnRHA. Changes in expression of seven randomly selected representative genes identified by the microarray technique were confirmed by real-time quantitative PCR and semi-quantitative reverse transcription/PCR (RT/PCR). Modulation of expression of genes by activin suggests that activin may mediate its effects through a variety of signaling pathways.

DNA microarrays for pathogen detection and characterisation

DNA microarrays have three main potential diagnostic uses in clinical microbiology: detection of known pathogens, pathogen typing and novel pathogen discovery. Although DNA microarray platforms offer the ability to screen for a large number of agents in parallel, sensitivity is dependent on the ability to obtain adequate amounts of pathogen nucleic acids from collected samples. In general, high levels of sensitivity require a PCR amplification step using specific primer sets, subsequently reducing the overall scope of the microarray assay. At present, relatively high costs, restricted sample throughput capabilities and validation difficulties are also major factors limiting the implementation of DNA microarray assays in diagnostic microbiology laboratories.

Optimized DNA microarray assay allows detection and genotyping of single PCR-amplifiable target copies

This study was conducted to determine the detection limit of an optimized DNA microarray assay for detection and species identification of chlamydiae. Examination of dilution series of a plasmid standard carrying the target sequence from Chlamydia trachomatis and genomic DNA of this organism revealed that a single PCR-amplifiable target copy was sufficient to obtain a specific hybridization pattern. This performance renders the test suitable for routine testing of clinical samples.

Study of hepatitis B virus gene mutations with enzymatic colorimetry-based DNA microarray

Objectives To establish a modified microarray method for detecting HBV gene mutations in the clinic. Design and methods Site-specific oligonucleotide probes were immobilized to microarray slides and hybridized to biotin-labeled HBV gene fragments amplified from two-step PCR. Hybridized targets were transferred to nitrocellulose membranes, followed by intensity measurement using BCIP/NBT colorimetry. Results HBV genes from 99 Hepatitis B patients and 40 healthy blood donors were analyzed. Mutation frequencies of HBV pre-core/core and basic core promoter (BCP) regions were found to be significantly higher in the patient group (42%, 40% versus 2.5%, 5%, P < 0.01). Compared with a traditional fluorescence method, the colorimetry method exhibited the same level of sensitivity and reproducibility. Conclusions An enzymatic colorimetry-based DNA microarray assay was successfully established to monitor HBV mutations. Pre-core/core and BCP mutations of HBV genes could be major causes of HBV infection in HBeAg-negative patients and could also be relevant to chronicity and aggravation of hepatitis B.

Chromosomal and genetic abnormalities in benign and malignant meningiomas using DNA microarray

Background: Meningioma is the commonest brain tumor and many genetic abnormalities, such as the loss of chromosome 22q and the mutation of NF2, have been reported.Methods: These classical abnormalities were detected using Southern blot, PCR, fluorescence in situ hybridization and comparative genomic hybridization, but these methods examine only very limited regions or limited mapping resolution of the tumor genome. In this study, we used DNA microarray assay, which detects numerous genetic abnormalities simultaneously and analyses a global assessment of molecular events in meningioma cells. We studied 31 meningiomas by GenoSensor Array 300 in order to detect the chromosomal aberrations and genetic abnormalities in the whole genome.Results: This study demonstrated not only classical chromosomal aberration, such as loss of chromosome 22q in 19 meningiomas (61.3%), but also new genetic characteristics of meningiomas, such as amplification of MSH2 in 16 meningiomas (51.6%), deletion of GSCL in 13 meningiomas (41.9%) and deletion of HIRA in seven meningiomas (22.6%).Conclusions: These results suggest that DNA microarray assay is useful in research for the genetic characters of meningiomas and understanding tumorigenesis.

Transcriptional Response ofEscherichia colito External Zinc

Transcriptional response of Escherichia coli to extracellular zinc was studied using DNA microarray and S1 mapping assays. Addition of external zinc induced the expression of zinc exporter ZntA and inhibited the expression of zinc importer ZnuC. In the continuous presence of zinc, ZnuC repression took place at lower zinc concentrations than ZntA induction. The microarray assay indicated that the addition of excess external zinc induces the expression of many genes that are organized in the regulon for cysteine biosynthesis, implying that cysteine plays a role in transient trapping of free zinc for maintenance of zinc homeostasis. Besides the RpoE regulon, other genes were also induced by zinc, suggesting that periplasmic proteins denatured by zinc induce the genes for protein repair. The microarray data of the newly identified zinc-responsive promoters were confirmed by S1 mapping.

The extract of huanglian, a medicinal herb, induces cell growth arrest and apoptosis by upregulation of interferon-β and TNF-α in human breast cancer cells

Huanglian (Coptidis rhizoma), a widely used herb in traditional Chinese medicine, has been shown recently to possess anticancer activities. However, the molecular mechanism underlying the anticancer effect of the herb is poorly understood. Specifically, whether huanglian extract affects the expression of cancer-related genes has not been defined. This study used DNA microarray technology to examine the effect of the herbal extract on expression of the common genes involved in carcinogenesis in two human breast cancer cell lines, the ER-positive MCF-7 and ER-negative MDA-MB-231 cells. Treatment of the cancer cells with huanglian extract markedly inhibited their proliferation in a dose- and time-dependent manner. The growth inhibitory effect was much more profound in MCF-7 cell line than that in MDA-MB-231 cells. DNA microarray assay revealed that treatment with huanglian dramatically increased the mRNA expression of interferon-beta (IFN-beta) and tumor necrosis factor-alpha in MCF-7 cells. Quantitative analysis by real-time PCR or western blotting confirmed the upregulation of the two genes (especially IFN-beta) in MCF-7 cells, but not in MDA-MB-231 cells. Addition of neutralizing antibody against IFN-beta to culture medium markedly inhibited the huanglian-induced antiproliferative effect, confirming the involvement of IFN-beta in the huanglian's effect and also suggesting an autocrine pathway for the action of IFN-beta in this setting. Given that IFN-beta is among the most important anticancer cytokines, the upregulation of this gene by huanglian is, at least in part, responsible for its antiproliferative effect. The results of this study implicate huanglian as a promising herb for chemoprevention and chemotherapy of certain cancers.

An analysis of the use of genomic DNA as a universal reference in two channel DNA microarrays

BackgroundDNA microarray is an invaluable tool for gene expression explorations. In the two-dye microarray, fluorescence intensities of two samples, each labeled with a different dye, are compared after hybridization. To compare a large number of samples, the 'reference design' is widely used, in which all RNA samples are hybridized to a common reference. Genomic DNA is an attractive candidate for use as a universal reference, especially for bacterial systems with a low percentage of non-coding sequences. However, genomic DNA, comprising of both the sense and anti-sense strands, is unlike the single stranded cDNA usually used in microarray hybridizations. The presence of the antisense strand in the 'reference' leads to reactions between complementary labeled strands in solution and may cause the assay result to deviate from true values.ResultsWe have developed a mathematical model to predict the validity of using genomic DNA as a reference in the microarray assay. The model predicts that the assay can accurately estimate relative concentrations for a wide range of initial cDNA concentrations. Experimental results of DNA microarray assay using genomic DNA as a reference correlated well to those obtained by a direct hybridization between two cDNA samples. The model predicts that the initial concentrations of labeled genomic DNA strands and immobilized strands, and the hybridization time do not significantly affect the assay performance. At low values of the rate constant for hybridization between immobilized and mobile strands, the assay performance varies with the hybridization time and initial cDNA concentrations. For the case where a microarray with immobilized single strands is used, results from hybridizations using genomic DNA as a reference will correspond to true ratios under all conditions.ConclusionSimulation using the mathematical model, and the experimental study presented here show the potential utility of microarray assays using genomic DNA as a reference. We conclude that the use of genomic DNA as reference DNA should greatly facilitate comparative transcriptome analysis.