The antioxidant tempol transforms gut microbiome to resist obesity in female C3H mice fed a high fat diet.

The nitroxide, Tempol, prevents obesity related changes in mice fed a high fat diet (HFD). The purpose of this study was to gain insight into the mechanisms that result in such changes by Tempol in female C3H mice. Microarray methodology, Western blotting, bile acid analyses, and gut microbiome sequencing were used to identify multiple genes, proteins, bile acids, and bacteria that are regulated by Tempol in female C3H mice on HFD. The effects of antibiotics in combination with Tempol on the gut microflora were also studied. Adipose tissue, from Tempol treated mice, was analyzed using targeted gene microarrays revealing up-regulation of fatty acid metabolism genes (Acadm and Acadl > 4-fold, and Acsm3 and Acsm5 > 10-fold). Gene microarray studies of liver tissue from mice switched from HFD to Tempol HFD showed down-regulation of fatty acid synthesis genes and up-regulation of fatty acid oxidation genes. Analyses of proteins involved in obesity revealed that the expression of aldehyde dehydrogenase 1A1 (ALDH1A1) and fasting induced adipose factor/angiopoietin-like protein 4 (FIAF/ANGPTL4) was altered by Tempol HFD. Bile acid studies revealed increases in cholic acid (CA) and deoxycholic acid (DCA) in both the liver and serum of Tempol treated mice. Tempol HFD effect on the gut microbiome composition showed an increase in the population of Akkermansia muciniphila, a bacterial species known to be associated with a lean, anti-inflammatory phenotype. Antibiotic treatment significantly reduced the total level of bacterial numbers, however, Tempol was still effective in reducing the HFD weight gain. Even after antibiotic treatment Tempol still positively influenced several bacterial species such as as Akkermansia muciniphila and Bilophila wadsworthia. The positive effects of Tempol moderating weight gain in female mice fed a HFD involves changes to the gut microbiome, bile acids composition, and finally to changes in genes and proteins involved in fatty acid metabolism and storage.

Demonstration of acid and water recovery systems: Applicability and operational challenges in Indian metal finishing SMEs.

Diffusion dialysis, acid retardation and nanofiltration plants were acquired from Europe and demonstrated in several Indian metal finishing companies over a three year period. These companies are primarily small and medium enterprises (SMEs). Free acid recovery rate from spent pickling baths using diffusion dialysis and retardation was in the range of 78-86% and 30-70% respectively. With nanofiltration, 80% recovery rate of rinse water was obtained. The demonstrations created awareness among the metal finishing companies to reuse resources (acid/water) from the effluent streams. However, lack of efficient oil separators, reliable chemical analysis and trained personnel as well as high investment cost limit the application of these technologies. Local manufacturing, plant customization and centralized treatment are likely to encourage the uptake of such technologies in the Indian metal finishing sector.

Design, synthesis and evaluation of novel pyrazolo-pyrimido[4,5-d]pyrimidine derivatives as potent antibacterial and biofilm inhibitors.

An efficient four-component reaction of 6-amino-1,3-dimethyluracil, N,N-dimethylformamide dimethylacetal, 1-phenyl-3-(4-substituted-phenyl)-4-formyl-1H-pyrazoles and aromatic amines was conducted in the presence of [Bmim]FeCl4 ionic liquid as a promoting medium. This strategy provided a convenient route without any additional catalyst or metal salt under mild conditions. All the synthesized pyrazolo-pyrimido[4,5-d]pyrimidines derivatives were evaluated for their antibacterial, minimum bactericidal concentration (MBC), biofilm inhibition, intracellular ROS accumulation and protein leakage activities. The results revealed that among all the screened derivatives, the compounds 5c, 5i, 5l and 5m were quite promising with MIC values ranging between 3.9 and 15.6µg/mL, while the MBC values were 2-fold the antibacterial activity values. The biofilm inhibition activity revealed that the compounds 5l and 5m exhibited promising activity with IC50 values ranging between 1.8 and 8.2µg/mL. It was observed that at a concentration of 0.5µg/mL, the compound 5l treated biofilms of Micrococcus luteus showed increased levels of intracellular ROS accumulation. Further, the protein leakage study revealed that the Micrococcus luteus cells treated with compound 5l caused membrane permeability which resulted in protein leakage and subsequent bacterial cell death.

An efficient one-pot synthesis of thiochromeno[3,4-d]pyrimidines derivatives: Inducing ROS dependent antibacterial and anti-biofilm activities.

An efficient synthesis of thiochromeno[3,4-d]pyrimidine derivatives has been achieved successfully via a one-pot three-component reaction of thiochrome-4-one, aromatic aldehyde and thiourea in the presence of 1-butyl-3-methyl imidazolium hydrogen sulphate [Bmim]HSO4. This new protocol has the advantages of environmental friendliness, high yields, short reaction times, and convenient operation. Furthermore, among all the tested derivatives, compounds 4b and 4c exhibited promising antibacterial, minimum bactericidal concentration and anti-biofilm activities against Staphylococcus aureus MTCC 96, Staphylococcus aureus MLS16 MTCC 2940 and Bacillus subtilis MTCC 121. The compound 4c also showed promising intracellular ROS accumulation in Staphylococcus aureus MLS16 MTCC 2940 comparable to that of ciprofloxacin resulting in apoptotic cell death of the bacterium.

Ionic liquid-promoted multicomponent synthesis of fused tetrazolo[1,5-a]pyrimidines as α-glucosidase inhibitors.

A simple and facile synthesis of fused tetrazolo[1,5-a]pyrimidine derivatives based on the multicomponent reaction of acetophenone, dimethylformamidedimethylacetal and 5-aminotetrazole is described. A green chemical synthesis has been achieved by using1-butyl-3-methylimidazolium hydrogen sulfate [Bmim]HSO4 ionic liquid as a reusable medium. Short synthetic route, operational simplicity, good yields, eco-friendliness and recyclability of the ionic liquid are the advantages of this method. The synthesized compounds were screened for α-glucosidase inhibitory activity using yeast maltase (MAL12) as a model enzyme. Inhibition and kinetic studies have shown that compounds 4d and 4g are found to be active showing comparable inhibitory potency with acarbose. Further docking studies of the derivatives with MAL12 homology model identified a similar binding mode consistent with the binding of acarbose. These studies along with in silico predicted ADMET properties suggest that these molecules could represent a new scaffold that may be useful for the development of new anti-diabetic drugs.

An expeditious four-component domino protocol for the synthesis of novel thiazolo[3,2-a]thiochromeno[4,3-d]pyrimidine derivatives as antibacterial and antibiofilm agents.

An efficient domino protocol has been developed for the synthesis of new pyrimidine scaffolds, through a one-pot four-component cascade transformation via [Bmim]HSO4 ionic liquid mediated reaction, using an equimolar mixture of thiochroman-4-one, benzaldehyde, thiourea and 3-bromo-1-phenylpropan-1-one leading to the formation of a double electrophilic pyrimidine-2(5H)-thione intermediate. The intermediate regioselectively undergoes cyclization through intramolecular NH bond activation followed by CS bond formation leading to highly functionalized thiazolo[3,2-a]thiochromeno[4,3-d]pyrimidines. The ionic liquid operates efficiently under mild conditions. The recyclability and scope for recovery of the ionic liquid makes this protocol environmentally benign. Further, the compounds 5d, 5g and 5k showed promising antimicrobial activity against the tested Gram-positive bacterial strains. Among them, the compound 5d was identified as a lead molecule exhibiting promising anti-biofilm activity towards Staphylococcus aureus MTCC 96, Bacillus subtilis MTCC 121, Staphylococcus aureus MLS16 MTCC 2940 and Micrococcus luteus MTCC 2470 with IC50 values of 2.1, 1.9, 2.4 and 5.3µg/mL, respectively. Further, the compound 5d showed increased levels of intracellular ROS accumulation in Staphylococcus aureus MTCC 96 suggesting that oxidative stress resulted in bacterial cell lysis and death.

Altered expression of microRNA-451 in eutopic endometrium of baboons (Papio anubis) with endometriosis.

STUDY QUESTION: Are microRNAs (miRs) altered in the eutopic endometrium (EuE) of baboons following the induction of endometriosis? SUMMARY ANSWER: Induction of endometriosis causes significant changes in the expression of eight miRs, including miR-451, in the baboon endometrium as early as 3 months following induction of the disease. WHAT IS KNOWN ALREADY: Endometriosis is one of the most common gynecological disorders and causes chronic pelvic pain and infertility in women of reproductive age. Altered expression of miRs has been reported in women and has been suggested to play an important role in the pathophysiology of several gynecological disorders including endometriosis. STUDY DESIGN, SIZE, DURATION: EuE was obtained from the same group of baboons before and 3 months after the induction of endometriosis. The altered expression of miR-451 was validated in the eutopic and ectopic endometrium of additional baboons between 3 and 15 months following disease induction. Timed endometrial biopsies from women with and without endometriosis were also used to validate the expression of miR-451. PARTICIPANTS/MATERIALS, SETTING, METHODS: Total RNA was extracted from EuE samples before and after the induction of endometriosis, and miRNA expression was analyzed using a 8 × 15 K miR microarray. Microarray signal data were preprocessed by AgiMiRna software, and an empirical Bayes model was used to estimate the changes. The present study focused on quantitative RT-PCR validation of the microarray data, specifically on miR-451 and its target genes in both baboons (n = 3) and women [control (n = 7) and endometriosis (n = 19)]. Descriptive and correlative analysis of miR-451 and target gene expression was conducted using in situ hybridization and immunohistochemistry, while functional analysis utilized an in vitro 3' untranslated region (UTR) luciferase assay and overexpression of miR-451 in human endometrial and endometriotic cell lines. MAIN RESULTS AND THE ROLE OF CHANCE: Induction of endometriosis results in the altered expression of miR-451, -141, -29c, -21, -424, -19b, -200a and -181a in the baboon endometrium. In the baboon, induction of endometriosis significantly decreased the expression of miR-451 at 3 months (P < 0.001), which was also associated with increased expression of its target gene YWHAZ (14.3.3ζ). A similar significant (P < 0.0001) decrease in miR-451 expression was observed in women with endometriosis. The 3' UTR luciferase assay confirmed the regulation of YWHAZ expression by miR-451. Furthermore, overexpression of miR-451 in 12Z cells (immortalized human endometriotic epithelial cell line) led to the decreased expression of its target YWHAZ and this was correlated with decreased cell proliferation. LIMITATIONS, REASONS FOR CAUTION: The study focused only on miR-451 and one of its targets, namely YWHAZ. A single miR could target number of genes and a single gene could also be regulated by number of miRs; hence, it is possible that other miRs and their regulated genes may contribute to the pathophysiology of endometriosis. WIDER IMPLICATIONS OF THE FINDINGS: Our data suggest that the presence of ectopic lesions in baboon causes changes in EuE miR expression as early as 3 months postinduction of the disease, and some of these changes may persist throughout the course of the disease. We propose that the marked down-regulation of miR-451 in both baboons and women with endometriosis increases the expression of multiple target genes. Increased expression of one of the target genes, YWHAZ, increases proliferation, likely contributing to the pathophysiology of the disease.

One-pot three-component domino protocol for the synthesis of novel pyrano[2,3-d]pyrimidines as antimicrobial and anti-biofilm agents.

A simple and facile synthesis of a series of novel pyrano[2,3-d]pyrimidines has been achieved successfully via the one-pot three-component reaction of 2-amino-7-methyl-5-oxo-4-phenyl-4,5-dihydropyrano[4,3-b]pyran-3-carbonitriles, DMF-DMA and arylamines in the presence of 1-butyl-3-methylhydrogensulphate [Bmim]HSO4 ionic liquid. This method has several advantages such as high yields, clean reaction, simple methodology and short reaction times. The synthesized compounds were evaluated for their antimicrobial activity against Gram-positive, Gram-negative and different Candida strains. Among the derivatives screened, compounds 4c, 4d, 4h and 4l were found to be active against both bacterial and Candida strains with MIC values ranging from 3.9 to 31.2 µg mL(-1). In addition, compound 4l showed a good minimum bactericidal concentration, minimum fungicidal concentration and anti-biofilm activities. Furthermore, the mode of the antifungal action for the promising compound 4l was evaluated in C. albicans MTCC 1637 through an ergosterol biosynthesis inhibition process.

Design, Synthesis, and In Vitro Antimicrobial Evaluation of Fused Pyrano[3,2-e]tetrazolo[1,5-c]pyrimidines and Diazepines.

A series of novel pyranochromene-containing tetrazoles fused with pyrimidinethiones, pyrimidines, and diazepines 3a-f, 4a-f, and 5a-f were synthesized by condensation of the corresponding tetrazoles 2a-f with carbon disulfide, benzaldehyde, and 4-methoxy phenacyl bromide, respectively. The compound 2a-f was obtained by reaction of pyrano[3,2-c]chromenes 1a-f with sodium azide. The structures of the newly synthesized compounds 2a-f to 5a-f were established on the basis of their elemental analyses, IR, (1)H NMR, (13)C NMR, and mass spectral data. All of the title compounds were subjected to in vitro antibacterial testing against four pathogenic strains and antifungal screening against two fungi. Preliminary results indicate that some of them exhibited promising activities and that they deserve more consideration as potential antimicrobials.

Identification of new Rel/NFkappaB regulatory networks by focused genome location analysis.

NFkappaB is an inducible transcription factor that controls kinetically complex patterns of gene expression. Several studies reveal multiple pathways linking NFkappaB to the promotion and progression of various cancers. Despite extensive interest and characterization, many NFkappaB controlled genes still remain to be identified. We used chromatin immunoprecipitation combined with microarray technology (ChIP/chip) to investigate the dynamic interaction of NFkappaB with the promoter regions of 100 genes known to be expressed in mitogen-induced T-cells. Six previously unrecognized NFkappaB controlled genes (ATM, EP300, TGFbeta, Selectin, MMP-1 and SFN) were identified. Each gene is induced in mitogen-stimulated T-cells, repressed by pharmacological NFkappaB blockade, reduced in cells deficient in the p50 NFkappaB subunit and dramatically repressed by RNAi specifically designed against cRel. A coregulatory role for Ets transcription factors in the expression of the NFkappaB controlled genes was predicted by comparative promoter analysis and confirmed by ChIP and by functional disruption of Ets. NFkappaB deficiency produces a deficit in ATM function and DNA repair indicating an active role for NFkappaB in maintaining DNA integrity. These results define new potential targets and transcriptional networks governed by NFkappaB and provide novel functional insights for the role of NFkappaB in genomic stability, cell cycle control, cell-matrix and cell-cell interactions during tumor progression.

Gene expression in human oral squamous cell carcinoma is influenced by risk factor exposure.

Oral squamous cell carcinoma (OSCC) is a world health problem and is associated with exposure to different risk factors. In the west, smoking and alcohol consumption are considered to be the main risk factors whilst in India and southeast Asia, betel quid (BQ) chewing is predominant. In this study, we compared the gene expression patterns of oral cancers associated with BQ chewing to those caused by smoking using Affymetrix microarrays. We found that 281 genes were differentially expressed between OSCC and normal oral mucosa regardless of aetiological factors including MMP1, PLAU, MAGE-D4, GNA12, IFITM3 and NMU. Further, we identified 168 genes that were differentially expressed between the BQ and smoking groups including CXCL-9, TMPRSS2, CA12 and RNF24. The expression of these genes was validated using qPCR using independent tissue samples. The results demonstrate that whilst common genes/pathways contribute to the development of oral cancer, there are also other gene expression changes that are specific to certain risk factors. The findings suggest that different carcinogens activate or inhibit specific pathways during cancer development and progression. These unique gene expression profiles should be taken into consideration when developing biomarkers for future use in prognostic or therapeutic applications.

Overexpression of folate binding protein and mesothelin are associated with uterine serous carcinoma.

PURPOSE: Folate receptor alpha (FOLR1) is a membrane bound receptor involved in the transport of folate as well as other regulatory cellular processes. The purpose of this study was to examine the expression of FOLR1 in uterine cancers and to identify changes in gene expression that are associated with overexpression of FOLR1. EXPERIMENTAL DESIGN: Fifty-eight frozen uterine cancer specimens were stained for FOLR1 using immunohistochemistry and results were correlated with transcript expression noted on quantitative PCR. Total RNA from 16 cases of uterine serous carcinoma (USC) was analyzed for gene expression using the Affymetrix HG-U133A and HG-U133B GeneChip set. USCs overexpressing FOLR1 were compared to cancers with an absence of FOLR1 using binary comparison and template matching of data was used to identify genes that correlate with FOLR1 expression. Selected targets from this analysis were evaluated by quantitative PCR as well as in an independent set of USC represented in quadruplicate on a tissue microarray (TMA). RESULTS: Overexpression of FOLR1 was observed in 11/16 (69%) of USC and 0/10 normal endometrium cases using frozen tissue specimens. Binary comparison between FOLR1 positive and negative cases identified 121 genes altered by 2-fold at p<0.01 of which 45 are well correlated with FOLR1 expression pattern. Using quantitative PCR, both mesothelin (MSLN) and PTGS1 (COX1) were significantly increased in FOLR1 overexpressing tumors (p=0.014 and p=0.006 respectively). TMA confirmed that overexpression of FOLR1 and MSLN respectively occurred in 23/48 (48%) and 17/54 (32%) of pure USC. CONCLUSION: Both FOLR1 and MSLN are cell surface targets that are co-expressed at high levels in USC and are appealing targets for biologic therapy.

Gene expression analysis of tumor infiltrating lymphocyte markers in endometrial cancers indicates no significant increases in those cases with microsatellite instability.

Microsatellite instability (MSI) is seen in many cancers and is the result of either a germline or somatic defect in the DNA mismatch repair system. Microsatellite instability is common in endometrial cancers occurring in about 25% of cases with endometrioid histology. Tumor infiltrating lymphocytes (TIL) are more prominent in colorectal cancer cases with MSI. The presence of increased TIL is associated with increased survival in these colorectal cancers, and is suggested as one possible mechanism to explain the increased survival rates in colorectal cancer patients with MSI positive cancers. Some degree of evidence indicates that increased TIL is also predictive of increased survival in endometrial cancer. The relative levels and states of activation of TIL in endometrial cancers with and without MSI has not been explored. Our previous data indicates that global gene expression patterns from MSI and non-MSI endometrial cancers are distinct, however TIL markers were not over-represented on statistically relevant gene lists that distinguish these groups. We further examined these pre-existing microarray data by directly querying transcripts present in the T-cell gene ontology (GO) group. No significant differences were observed between MSI and microsatellite stable (MSS) groups. Finally we directly examined a set of T-cell marker transcripts previously utilized to define increased activated and cytotoxic TIL in MSI positive colorectal cancers. Whereas colorectal cancers with MSI have been previously demonstrated to contain higher ratios of CD8/CD3 message levels we observed no difference in endometrial cancers. In addition, levels of CD3 indicated no increases in TIL in MSI positive cases and 2 markers of activation, granzyme B and IL-2R were not different in MSI positive and negative cancers. These data indicate that significant differences in TIL derived transcripts do not occur between endometrioid endometrial cancers with and without microsatellite instability.

Selective leukemic-cell killing by a novel functional class of thalidomide analogs.

Using a novel cell-based assay to profile transcriptional pathway targeting, we have identified a new functional class of thalidomide analogs with distinct and selective antileukemic activity. These agents activate nuclear factor of activated T cells (NFAT) transcriptional pathways while simultaneously repressing nuclear factor-kappaB (NF-kappaB) via a rapid intracellular amplification of reactive oxygen species (ROS). The elevated ROS is associated with increased intracellular free calcium, rapid dissipation of the mitochondrial membrane potential, disrupted mitochondrial structure, and caspase-independent cell death. This cytotoxicity is highly selective for transformed lymphoid cells, is reversed by free radical scavengers, synergizes with the antileukemic activity of other redox-directed compounds, and preferentially targets cells in the S phase of the cell cycle. Live-cell imaging reveals a rapid drug-induced burst of ROS originating in the endoplasmic reticulum and associated mitochondria just prior to spreading throughout the cell. As members of a novel functional class of "redoxreactive" thalidomides, these compounds provide a new tool through which selective cellular properties of redox status and intracellular bioactivation can be leveraged by rational combinatorial therapeutic strategies and appropriate drug design to exploit cell-specific vulnerabilities for maximum drug efficacy.

Transcriptional responses to ionizing radiation reveal that p53R2 protects against radiation-induced mutagenesis in human lymphoblastoid cells.

The p53 protein has been implicated in multiple cellular responses related to DNA damage. Alterations in any of these cellular responses could be related to increased genomic instability. Our previous study has shown that mutations in p53 lead to hypermutability to ionizing radiation. To investigate further how p53 is involved in regulating mutational processes, we used 8K cDNA microarrays to compare the patterns of gene expression among three closely related human cell lines with different p53 status including TK6 (wild-type p53), NH32 (p53-null), and WTK1 (mutant p53). Total RNA samples were collected at 1, 3, 6, 9, and 24 h after 10 Gy gamma-irradiation. Template-based clustering analysis of the gene expression over the time course showed that 464 genes are either up or downregulated by at least twofold following radiation treatment. In addition, cluster analyses of gene expression profiles among these three cell lines revealed distinct patterns. In TK6, 165 genes were upregulated, while 36 genes were downregulated. In contrast, in WTK1 75 genes were upregulated and 12 genes were downregulated. In NH32, only 54 genes were upregulated. Furthermore, we found several genes associated with DNA repair namely p53R2, DDB2, XPC, PCNA, BTG2, and MSH2 that were highly induced in TK6 compared to WTK1 and NH32. p53R2, which is regulated by the tumor suppressor p53, is a small subunit of ribonucleotide reductase. To determine whether it is involved in radiation-induced mutagenesis, p53R2 protein was inhibited by siRNA in TK6 cells and followed by 2 Gy radiation. The background mutation frequencies at the TK locus of siRNA-transfected TK6 cells were about three times higher than those seen in TK6 cells. The mutation frequencies of siRNA-transfected TK6 cells after 2 Gy radiation were significantly higher than the irradiated TK6 cells without p53R2 knock down. These results indicate that p53R2 was induced by p53 protein and is involved in protecting against radiation-induced mutagenesis.

Pharmacologic profiling of transcriptional targets deciphers promoter logic.

The blueprint for cellular diversity and response to environmental change is encoded in the cis-acting regulatory sequences of most genes. Deciphering this 'cis-regulatory code' requires multivariate data sets that examine how these regions coordinate transcription in response to diverse environmental stimuli and therapeutic treatments. We describe a transcriptional approach that profiles the activation of multiple transcriptional targets against combinatorial arrays of therapeutic and signal transducing agents. Application of this approach demonstrates how cis-element composition and promoter context combine to influence transcription downstream of mitogen-induced signaling networks. Computational dissection of these transcriptional profiles in activated T cells uncovers a novel regulatory synergy between IGF-1 and CD28 costimulation that modulates NF-kappaB and AP1 pathways through signaling cascades sensitive to cyclosporin A and wortmannin. This approach provides a broader view of the hierarchical signal integration governing gene expression and will facilitate a practical design of combinatorial therapeutic strategies for exploiting critical control points in transcriptional regulation.

Microarray analysis of endometrial carcinomas and mixed mullerian tumors reveals distinct gene expression profiles associated with different histologic types of uterine cancer.

Previous studies using cDNA microarray have indicated that distinct gene expression profiles characterize endometrioid and papillary serous carcinomas of the endometrium. Molecular studies have observed that mixed mullerian tumors, characterized by both carcinomatous and sarcomatous components, share features that are characteristic of endometrial carcinomas. The objective of this analysis was to more precisely define gene expression patterns that distinguish endometrioid and papillary serous histologies of endometrial carcinoma and mixed mullerian tumors of the uterus. One hundred nineteen pathologically confirmed uterine cancer samples were studied (66 endometrioid, 24 papillary serous, and 29 mixed mullerian tumors). Gene expressions were analyzed using the Affymetrix Human Genome Arrays U133A and U133B Genechip set. Unsupervised analysis revealed distinct global gene expression patterns of endometrioid, papillary serous, mixed mullerian tumors, and normal tissues as grossly separated clusters. Two-sample t tests comparing endometrioid and papillary serous, endometrioid and mixed mullerian tumor, and papillary serous and mixed mullerian tumor pairs identified 1,055, 5,212, and 1,208 differentially expressed genes at P < 0.001, respectively. These data revealed that distinct patterns of gene expression characterize various histologic types of uterine cancer. Gene expression profiles for select genes were confirmed using quantitative PCR. An understanding of the molecular heterogeneity of various histologic types of endometrial cancer has the potential to lead to better individualization of treatment in the future.

Three microarray platforms: an analysis of their concordance in profiling gene expression.

BACKGROUND: Microarrays for the analysis of gene expression are of three different types: short oligonucleotide (25-30 base), long oligonucleotide (50-80 base), and cDNA (highly variable in length). The short oligonucleotide and cDNA arrays have been the mainstay of expression analysis to date, but long oligonucleotide platforms are gaining in popularity and will probably replace cDNA arrays. As part of a validation study for the long oligonucleotide arrays, we compared and contrasted expression profiles from the three formats, testing RNA from six different cell lines against a universal reference standard. RESULTS: The three platforms had 6430 genes in common. In general, correlation of gene expression levels across the platforms was good when defined by concordance in the direction of expression difference (upregulation or downregulation), scatter plot analysis, principal component analysis, cell line correlation or quantitative RT-PCR. The overall correlations (r values) between platforms were in the range 0.7 to 0.8, as determined by analysis of scatter plots. When concordance was measured for expression ratios significant at p-values of <0.05 and at expression threshold levels of 1.5 and 2-fold, the agreement among the platforms was very high, ranging from 93% to 100%. CONCLUSION: Our results indicate that the long oligonucleotide platform is highly suitable for expression analysis and compares favorably with the cDNA and short oligonucleotide varieties. All three platforms can give similar and reproducible results if the criterion is the direction of change in gene expression and minimal emphasis is placed on the magnitude of change.

Evaluation of hybridization conditions for spotted oligonucleotide-based DNA microarrays.

We compared different hybridization conditions of oligonucleotide-based DNA microarray to acquire optimized and reliable microarray data. Several parameters were evaluated at different hybridization conditions, including signal-to-background (S:B) ratios, signal dynamic range, usable spots, and reproducibility. Statistical analysis showed that better results were obtained when spotted, presynthesized long oligonucleotide arrays were blocked with succinic anhydride and hybridized at 42 degrees C in the presence of 50% formamide.