Relative Gene Expression Values Research Articles

Optimization Approaches for the In Silico Discovery of Optimal Targets for Gene Over/Underexpression

Metabolic engineering (ME) efforts have been recently boosted by the increase in the number of annotated genomes and by the development of several genome-scale metabolic models for microbes of interest in industrial biotechnology. Based on these efforts, strain optimization methods have been proposed to reach the best set of genetic changes to apply to selected host microbes, in order to create strains that are able to overproduce metabolites of industrial interest. Previous work in strain optimization has been mostly based in finding sets of gene (or reaction) deletions that lead to desired phenotypes in computational simulations. In this work, we focus on enlarging the set of possible genetic changes, considering gene over and underexpression. A gene is considered under (over) expressed if its expression value is constrained to be significantly lower (higher) than the one in the wild-type strain, used as a reference. A method is proposed to propagate relative gene expression values to flux constraints over related reactions, making use of the available transcriptional/translational information. The algorithms chosen for the optimization tasks are metaheuristics such as Evolutionary Algorithms (EA) and Simulated Annealing (SA), based on previous successful work on gene knockout optimization. These methods were modified appropriately to accommodate the novel optimization tasks and were applied to study the optimization of succinic and lactic acid production using Escherichia coli as the host. The results are compared with previous ones obtained in gene knockout optimization, thus showing the usefulness of the approach. The methods proposed in this work were implemented in a novel plug-in for OptFlux, an open-source software framework for ME. Supplementary Material is available at www.liebertonline.com/cmb.

Extraction of RNA Using Fine-Needle Aspiration Samples Stored Under Different Conditions

: Diagnosis of lung cancer is achieved by fine-needle aspiration (FNA) techniques such as computed tomography-guided FNA and transbronchial needle aspiration. The purpose of this study was to establish an optimal storing method for molecular testing in FNA samples. : We performed FNA using a 21-gauge needle in surgically resected lung cancer samples. The aspirates were stored according to the following protocol: in group 1, the aspirate was snap frozen with liquid nitrogen and in group 2, the aspirate was mixed with RNA later. After sample collection from both groups, these samples were stored at -80°C for 6 months. RNA was extracted from each sample using a commercially available RNA extraction kit, and the quality and quantity of RNA were measured. Quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR) was performed for human actin β (hACTB) and keratin 19 (KRT19). : FNA was performed from 7 lung cancers. RNA was extracted from all samples. The median total amount of extracted RNA was 33.9 μg for group 1 and 35.8 μg for group 2. The mean RNA integrity number was 3.5 for group 1 and 6.3 for group 2. RT-PCR for hACTB and KRT19 could be successfully performed in all samples; however, the relative gene expression value showed intrasample variation. : Samples obtained from computed tomography-guided FNA or transbronchial needle aspiration may be used for genetic profiling of lung cancer. RNA can be extracted from FNA samples and can be used for RT-PCR. RNA quality may affect mRNA expression analysis; therefore, an optimal FNA sample storing protocol is essential.

Predicting Metabolic Fluxes Using Gene Expression Differences As Constraints

A standard approach to estimate intracellular fluxes on a genome-wide scale is flux-balance analysis (FBA), which optimizes an objective function subject to constraints on (relations between) fluxes. The performance of FBA models heavily depends on the relevance of the formulated objective function and the completeness of the defined constraints. Previous studies indicated that FBA predictions can be improved by adding regulatory on/off constraints. These constraints were imposed based on either absolute or relative gene expression values. We provide a new algorithm that directly uses regulatory up/down constraints based on gene expression data in FBA optimization (tFBA). Our assumption is that if the activity of a gene drastically changes from one condition to the other, the flux through the reaction controlled by that gene will change accordingly. We allow these constraints to be violated, to account for posttranscriptional control and noise in the data. These up/down constraints are less stringent than the on/off constraints as previously proposed. Nevertheless, we obtain promising predictions, since many up/down constraints can be enforced. The potential of the proposed method, tFBA, is demonstrated through the analysis of fluxes in yeast under nine different cultivation conditions, between which approximately 5,000 regulatory up/down constraints can be defined. We show that changes in gene expression are predictive for changes in fluxes. Additionally, we illustrate that flux distributions obtained with tFBA better fit transcriptomics data than previous methods. Finally, we compare tFBA and FBA predictions to show that our approach yields more biologically relevant results.

Increases In Mirna-145 and Mirna-146a Expression In Patients with IPSS Lower-Risk Myelodysplastic Syndromes and Del(5q) Treated with Lenalidomide.

AbstractAbstract 3631 Introduction:The erythroid differentiation defect observed in 5q– syndrome has been attributed to the RPS14 gene located within the CDR of the long arm of chromosome 5. We have recently demonstrated that RPS14 expression increases during lenalidomide treatment. However, haploinsufficiency of RPS14, which encodes ribosomal protein S14, does not explain clonal dominance. The expression of miRNAs, miR-145 (5q33.1) and miR-146a (5q33.3), in CD34+ bone marrow (BM) cells of individuals with MDS with deletion of the long arm of chromosome 5 (del(5q)) is lower compared to normal controls (Starczynowski et al, Nature Medicine, 2010). miRNAs are small noncoding RNAs that post-transcriptionally repress specific messenger RNA targets through interaction with the 3′ untranslated region (UTR). Loss of noncoding transcripts encoding miRNAs within the CDR may result in haploinsufficiency by loss of inhibition of their targets. Concurrent loss of both miR-145 and miR-146a resulted in activation of innate immune signalling through elevated expression of their respective targets, TIRAP and TRAF6. Furthermore, knockdown of miR-145 and miR-146a or overexpression of TRAF6 in mouse HSPC (Hematopoietic stem and progenitor cells) recapitulated features of 5q– syndrome, such as bone marrow dysplasia, anemia and thrombocytosis. We present preliminary results of changes in miRNA expression in IPSS lower-risk MDS with del(5q) during treatment with lenalidomide. Methods:A prospective single-arm trial investigating the efficacy and safety of lenalidomide in 46 patients with MDS with del(5q) with/without additional cytogenetic abnormalities and Hb < 10 g/dL. Lenalidomide was administered orally at a starting dose of 10 mg/day for a maximum of 12 months. When necessary, dosing was reduced to 5 mg/day or 5 mg on alternate days. Bone marrow assessments were performed at baseline and every 3 months, thereafter. For the evaluation of miRNA-145 and miRNA-146a in patient samples, 300 ng/μl of miRNAs were isolated in each purified BM sample by using mirVana™ miRNA Isolation Kit-Ambion and TaqMan miRNA Array Analysis was performed to determine the expression of miRNAs (7900HT Sequence Detection System Applied Biosystems). Patient BM-miRNAs were calibrated with miRNAs from BM of healthy volunteer donors. It was assumed that BM expression value of each calibrator miRNA was 1 unit. RPS14 gene assays were performed using TaqMan® Low Density Array Fluidic card (TaqMan® Human Array, Applied Biosystems, Foster City, CA, USA) based on Applied Biosystems PRISM® 7900HT comparative ddCT method, according to the manufacturer's instructions. Target gene expression levels were measured in triplicate and normalized against the expression of the 18S housekeeping gene from a BM pool of normal, healthy subjects at all timepoints. Median relative gene expression values in MDS patients were compared to healthy subjects, set as a value of 1. Results:Four patients have been evaluated (1 M, 3 F; ages 65, 66, 73 and 76 years, respectively) at baseline and after 12 weeks. At baseline, 2 patients were RBC-transfusion dependent. One patient had one additional cytogenetic abnormality (+8 in 15% metaphases). All patients obtained an erythroid response by week 12: mean Hb values significantly increased from 8.4 ± 0.9 at baseline to 11.6 ± 0.9 g/dL (p=0.01). All patients obtained a cytogenetic response, 2 of which were complete. miRNA-145 and miRNA-146a expression were both low at baseline and significantly increased by week 12 (Table). Conclusions:Preliminary results confirm that, in IPSS lower-risk MDS with del(5q), miRNA-145 and miRNA-146a expression is low. Lenalidomide treatment is associated with erythroid responses and cytogenetic remissions concurrent with significant increases in miRNA-145 and miRNA-146a expression.Table 1miRNA 145 and 146a and RPS14 expression before and during lenalidomide treatment.CasesmiRNA 145miRNA 146aRPS14Baseline12 weeksBaseline12 weeksBaseline12 weeks10.0004.8630.9361.7000.3057.62520.8363.8470.0871.6540.7198.83530.9276.9470.0342.7800.7352.03340.0672.8300.0740.8970.6376.164Mean ± SD0.458 ± 0.4924.622 ± 1.7580.283 ± 0.4361.758 ± 0.7750.599 ± 0.2016.164 ± 2.963p-value0.0130.0490.035 Disclosures:Oliva:Celgene: Consultancy.

Bone Marrow Immunological Changes During Treatment with Lenalidomide In Low and Intermediate-1 Risk Myelodysplastic Syndromes with Del(5Q)

AbstractAbstract 2927 Background:Immunological changes have a primary role in the initiation and progression of myelodysplastic syndromes (MDS). Cytokine levels, such as IL-7 and IFN-gamma, are associated with lower-risk disease. Treatment with lenalidomide has proven efficacy in red blood cell (RBC) transfusion-dependent lower-risk MDS patients with del(5q). Lenalidomide exerts anti-angiogenic, anti-proliferative, and pro-erythropoietic effects; in particular, it has been shown that lenalidomide inhibits the proliferation and function of T regulatory cells (Tregs). Finally, MDS patients undergoing lenalidomide treatment experience erythroid responses and suppression of the del(5q) clone. Aims:In a multicenter Italian phase II trial to evaluate safety and efficacy of lenalidomide in primary MDS patients with del(5q) and Low- or Int-1 risk IPSS, we investigated changes in the transcription of cytokines and their receptors during treatment. Methods:The starting dose of lenalidomide was 10 mg p.o once daily on a continuous daily schedule for a maximum of 12 months. Bone marrow (BM) aspirates were obtained on study entry and every 12 weeks. Assays were performed using TaqMan® Low Density Array Fluidic card (TaqMan® Human Array, Applied Biosystems, Foster City, CA, USA) based on Applied Biosystems PRISM® 7900HT comparative ddCT method, according to the manufacturer's instructions. Target gene expression levels were measured in triplicate and normalized against the expression of the 18S housekeeping gene from a BM pool of normal, healthy subjects at all timepoints. Median relative gene expression values in MDS patients were compared to healthy subjects, set as a value of 1. Results:We report data obtained at baseline and after 12 weeks. Informed consent was obtained in all patients. Twenty-seven patients (5 M, 22 F) were evaluated at baseline and after 12 weeks. Mean age was 72 ± 9 years. Mean Hb level was 8.5 ± 0.9 g/dL and 16 patients were RBC transfusion -dependent (requiring at least 4 RBC transfusions in the preceding 2 months). Seven patients had additional cytogenetic abnormalities. Twenty-one patients (80%) experienced erythroid responses by week 12. Significant variations in gene expression of cytokines and receptors were observed during treatment. Genes significantly regulated during lenalidomide treatment (P < 0.05) are shown in the Table. In particular, FAS, IL-7 and FOXP3 gene were generally under-expressed at baseline and significantly increased after 12 weeks. Accordingly, IL7R was over-expressed in all patients at baseline and its expression was significantly reduced during treatment. Furthermore, IFN-gamma expression increased during therapy. Summary:The protein encoded by FAS gene is a member of the TNF-receptor superfamily and its interaction with its ligand leads to apoptosis. Interleukin (IL)-7 is an essential cytokine that promotes the proliferation and survival of B- and T-lymphocyte progenitors. The IL7R gene on chromosome 5 (5p13) codifies for the IL7 receptor, which blocks apoptosis during differentiation and activation of T lymphocytes. It functions, in part, through the induction of the expression of the antiapoptotic protein Bcl-2. The protein encoded by the FOXP3 gene is a member of transcriptional regulators. Defects in this gene are the cause of X-linked autoimmunity-immunodeficiency syndrome. The results of the present study indicate that lenalidomide may act through immunological changes. Further detailed analyses in these patients may provide new insights into the pathogenesis of MDS with del(5q) and the long-term effects of lenalidomide treatment on immunological changes in BM cells.Table:Changes in gene expression of bone marrow cytokines during lenalidomide treatment in MDS del5q patients.GenesBaseline gene expression Median (IQ range)Gene expression after 12 weeks Median (IQ range)p-valueFAS0.04ü(0.02ü–ü0.60)23.68ü(0.09ü–ü586.51)<0.0001Gamma-IFN0.69ü(0.23ü–ü7.86)45.23ü(9.87ü–ü1138.67)0.001IL-12A0.35ü(0.15ü–ü7.35)3.84ü(0.27ü–ü202.29)0.030IL-12B0.31ü(0.08ü–ü6.64)22.75ü(0.37ü–ü2.70)0.002IL-70.29ü(0.06ü–ü0.86)4.94ü(0.19ü–ü25.07)0.012Il-7R2305.10ü(1081.50ü–ü6261–73)0.12ü(0.00ü–ü2.80)<0.0001IRF70.12ü(0.04ü–ü0.97)7.98ü(0.25ü–ü1.42)0.005FOXP30.83ü(0.35ü–ü2.02)7.22ü(3.71ü–ü13.20)<0.0001 Disclosures:Oliva:Celgene: Consultancy.

Abstract 3488: In vitro activity of the IGF-1R inhibitor CP-751,871, as a single agent and in combination with paclitaxel, in lung and colon cell lines

Abstract Background: Signaling through the insulin-like growth factor 1 receptor (IGF-1R) has been implicated in the resistance to a number of clinically relevant anti-cancer agents. Objective: To assess the direct anti-tumour effects of the IGF-1R monoclonal antibody CP-751,871, as a single agent and in combination with paclitaxel, in lung and colon cancer cell lines. Methods: Four lung (A549, HCC78, H1299 and H460) and four colon (LS180, Colo205, HT29 and DLD1) cancer cell lines were selected. The experiments were designed to test the effects of simultaneous or sequential exposure to with CP-751,871 and/or paclitaxel. In the sequential arm, cells were pretreated with progressive doses of paclitaxel (from 0 to1000 nM) for 24 hours, drug removed by washing the cells twice, followed by the addition of cell culture medium plus CP-751,871 (50 μg/mL). After 48 hours of continuous presence of CP-751,871, cell growth was evaluated by WST-1 cell survival assays. In simultaneous treatment, cells were treated with both Paclitaxel (0-1000 nM) and CP-751,871 (50 μg/mL) for 48 hours. Flow cytometric analysis was used to estimate the effects on cell cycle and apoptosis. IGF-IR mRNA expression was determined by quantitative real-time PCR. Relative gene expression values were calculated by the ΔΔCt method. Results: No correlation between basal IGF-IR mRNA expression and response to CP-751,871 was observed. Flow cytometric analysis demonstrated that CP-751,871 enhanced cell cycle arrest at the G1/G0 checkpoint with minimal effects on apoptosis. Combined exposure to both CP-751,871 and Paclitaxel showed improved response in cell growth inhibition on HCC78, H1299, Colo205 and HT29 cell lines, and was statistically superior to Paclitaxel alone (p&amp;lt; 0.001) and CP-751,871 alone (p&amp;lt; 0.001). The results were even better after a sequential exposure. On the contrary, in H460, LS180 and DLD-1 cell lines, concomitant, but no sequential treatment resulted in antagonistic interactions. Conclusions: CP-751,871 showed a modest anti-tumour activity (about 30% growth inhibition in lung cancer cells; 10% growth inhibition in DLD1 and HT29 cell lines; and bigger inhibition in Colo205 (IC50=27.6 μg/mL) and LS180 (IC50=2.33 μg/mL) cell lines), predominantly cytostatic, that is not related to the mRNA expression. The effects of CP-751,871 in combination with paclitaxel depend on the cell line model and on the sequence of administration. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3488.

Evaluation on the IGF-IR Inhibitor CP-751,871, alone and in combination with paclitaxel in lung and colon cell lines

e22125 Background: Signaling through the insulin-like growth factor 1 receptor (IGF-IR) has been implicated in the resistance to a number of clinically relevant anti-cancer agents. The aim of this study was to assess the direct anti-tumour effects of CP-751,871, alone and in combination with paclitaxel in lung and colon cancer cell lines. Methods: Four lung and four colon cancer cell line where treated with the IGF-IR inhibitor CP-751,871 and/or Paclitaxel in simultaneous or sequential treatments. Response to treatments was evaluated by WST-1 cell survival assays. Flow cytometric analysis was used to estimate the effect on the cell cycle and apoptosis. IGF-IR mRNA expression was determined by quantitative real-time PCR and relative gene expression values were calculated by the ΔΔCt method. Results: No correlation between basal IGF-IR mRNA expression and CP-751,871 response was observed in cell lines tested. Flow cytometric analysis demonstrated that CP-751,871 enhanced cell cycle arrest at the G1/G0 checkpoint with minimal effects on apoptosis. Combined simultaneous and, more strongly, sequential treatment with CP- 751,871 and Paclitaxel showed improved response in cell growth inhibition on HCC78, H1299, Colo205 and HT29 cell lines, and was statistically superior to Paclitaxel alone (p&lt; 0.001) and CP-751,871 alone (p&lt; 0.001). In H460, LS180 and DLD-1 cell lines, concomitant, but no sequential treatment resulted in antagonistic interactions. Conclusions: CP-751,871 showed a modest anti-tumour activity, predominantly cytostatic, against lung and colon cancer cell lines, that is not related to the mRNA expression. CP-751,871 tends to enhance the effects of paclitaxel in vitro, depending on sequence of administration and on the model system used. No significant financial relationships to disclose.

Isolation of circulating tumour cells in patients with metastatic breast cancer by filter technology.

Abstract Abstract #5026 Introduction. Metastasis is the leading cause of cancer-related death. For several decades, numerous attempts have been made to develop reliable assays enabling the detection and enumeration of circulating tumor cells (CTC) in the peripheral blood. The LeukoLOCK™ Total RNA Isolation System (Ambion®) combines leukocyte depletion filter technology intended to isolate leukocytes from whole blood, with RNALater® as a means of stabilizing the RNA in the cells captured on the filter. We hypothesized that this innovative system would also allow for the isolation of carcinoma cells by filtration, because of their larger size in comparison to peripheral blood leukocytes. The RNA extracted from the filter device could thus be used for further quantification of CTCs by qRT-PCR.&amp;#x2028; Materials and methods. The CellSearch™ Circulating Tumor Cell Test (Veridex®) was used to enrich and enumerate CTCs in all of the blood samples. The CellSearch System™utilizes iron nano-particles (ferrofluid) conjugated to EpCAM to capture CTC. Immuno-fluorescent staining for cytokeratins (CK) 8,18,19 and the nucleus (DAPI) is used to positively identify intact CTC. CTC are defined as nuclear cells, expressing cytokeratins and lacking CD45 expression. Blood samples of 5 patients with known elevated CTC were screened again after filtration with the LeukoLOCK™filter device to ensure that CTC did not slip through the filter (Table 1). The cell count obtained with the Veridex system was compared in these 5 samples, with the quantification CK19 and mammaglobin (MAM) transcripts obtained from the cells retained by the filter. In a further study on 79 patients with metastatic breast cancer, CTCs were enumerated using LeukoLOCK™Total RNA Isolation System (Ambion®) as a means of RNA isolation and compared with the Veridex system. qRT-PCR was performed to detect the presence of CTC transcripts for CK19 in the total RNA obtained from the cells captured on the filter in all of the 79 cases.&amp;#x2028; Results. In only one sample (PAT113) could one CTC be detected in the filtered blood. The values obtained for RGE for both CK19 and MAM are in agreement with that observation.&amp;#x2028; &amp;#x2028; A significant correlation was found between CTC levels and qRT-PCR normalized relative gene expression (nRGE) values for CK19 originating from the total cell population captured by the LeukoLOCK™filter device (n=79, r=0.446, p&amp;lt;0.001).&amp;#x2028; Conclusion. The LeukoLOCK™ system does not only enrich for leukocytes but can be considered as a reliable system for the enrichment and stabilization of CTC in patients with MBC. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5026.

Comparison of 5-Fluorouracil-related Gene Expression Levels Between Adenocarcinomas and Squamous Cell Carcinomas of the Lung

A recent meta-analysis study showed that post-operative adjuvant chemotherapy with UFT, an oral combination drug composed of tegafur [prodrug of 5-fluorouracil (5-FU)] and uracil [inhibitor of dihydropyrimidine dehydrogenase (DPD)] was associated with improved survival in patients with lung adenocarcinomas, but not in those with lung squamous cell carcinomas. We investigated the 5-FU-related gene expression levels of thymidylate synthase (TS), DPD, thymidine phosphorylase (TP) and orotate phosphoribosyl transferase (OPRT) in resected tumor specimens from 51 patients with adenocarcinomas and 47 with squamous cell carcinomas using quantitative reverse transcription-PCR, and compared those levels between the two histological types. The relative gene expression values of TS, TP and OPRT were significantly lower in adenocarcinomas compared with squamous cell carcinomas, 1.60 +/- 0.86 versus 4.33 +/- 3.40 (P < 0.001), 0.84 +/- 0.52 versus 2.27 +/- 1.16 (P = 0.006) and 9.59 +/- 6.30 versus 16.94 +/- 12.04 (P < 0.001), respectively. The relative gene expression value of DPD was significantly greater in adenocarcinomas than those in squamous cell carcinomas, 2.33 +/- 1.22 versus 1.50 +/- 1.20 (P = 0.01). Lower expressions of TS and TP were observed more in adenocarcinomas (89.8%) than in squamous cell carcinomas (48.9%) (P < 0.001). These data may explain that post-operative adjuvant chemotherapy with UFT was associated with improved survival in stage I patients with adenocarcinoma, but less with squamous cell carcinoma.

Two new members of the Tetrahymena multi-stress-inducible metallothionein family: Characterization and expression analysis of T. rostrata Cd/Cu metallothionein genes

We report the cloning and characterization of two new metallothionein (MT) genes ( TrosMTT1 and TrosMTT2), isolated as cDNAs, from the ciliated protozoa Tetrahymena rostrata. The TrosMTT1 inferred protein has been identified as a CdMT and included into the 7a subfamily of Tetrahymena MTs, while TrosMTT2 has been identified as a CuMT (including it into 7b subfamily), due to its similarity to TpigMT-2 and its significant induction by copper. TrosMTT1 protein sequence reveals a remarkably regular and hierarchical modular organization, as it is known for other Tetrahymena CdMTs, showing a bi-modular structure. TrosMTT2 presents a structural organization based on CKCX 2–5CKC repeats, like it occurs in other Tetrahymena CuMTs, indicating that an evolutionary history based on intra-gene duplications might be also possible. Both are also multi-stress-inducible genes because they are induced by other heavy metals and stressors, as it has been shown by quantitative real-time RT-PCR. It is the first time that the gene expression of a putative Tetrahymena CuMT is analyzed by quantitative PCR, confirming it as a CuMT. These two new Tetrahymena MTs complete, at present, the actual view of this protein superfamily, and corroborate the unique features of ciliate MTs. Furthermore, both, a comparative analysis of relative gene expression values obtained by quantitative RT-PCR on other Tetrahymena MT genes and an analysis of the different Tetrahymena MTs based on the different Cys clusters of these proteins are carried out, which show an update view of Tetrahymena MT gene family.

Mammaglobin B expression in human endometrial cancer

Mammaglobin B (MGB-2) is an uteroglobin gene family member recently found highly differentially expressed in ovarian cancer by gene expression profiling. To evaluate its potential as a novel endometrial cancer biomarker, in this study we quantified and compared MGB-2 expression at messenger RNA and protein levels in endometrial tumors (endometrioid endometrial cancer [EEC]) with different grades of differentiation. MGB-2 expression was evaluated by real-time polymerase chain reaction (PCR) and immunohistochemistry (IHC) in fresh frozen biopsies and paraffin-embedded tissues derived from a total of 70 patients including 50 primary EEC and 20 normal endometria (NECs). High levels of MGB-2 gene expression were detected in 10 of 11 EEC G1 cases (91%), 16 of 17 EEC G2 cases (94%), and 6 of 22 EEC G3 cases (27%) by real-time PCR. In contrast, normal endometrial cells expressed low to negligible levels of MGB-2 by real-time PCR (P = 0.002 EEC vs NEC). Well- and moderately differentiated EECs overexpressed MGB-2 gene at significant higher levels when compared to NECs (P < 0.01). Pairwise differences between both G2 and G1 vs G3 cases for MGB-2 relative gene expression values were also statistically significant (G2 vs G3 P < 0.001, G1 vs G3 P = 0.016). MGB-2 protein expression was detected in 31 (86%) of 36 EEC and 0 of 5 atrophic NEC controls, while seven of eight (88%) of the proliferative/secretory/hyperplastic NECs focally expressed MGB-2 by IHC. MGB-2 is highly expressed in EEC, particularly in well- and moderately differentiated tumors, and may represent a novel molecular marker for EEC.

Systematic Approaches for Incorporating Control Spots and Data Quality Information to Improve Normalization of cDNA Microarray Data

Background: Normalization and data quality control are two important aspects in microarray data analysis. Proper normalization and data quality control ensure that intensity ratios provide meaningful and accurate measurement of relative gene expression values. Control spots such as spikes and housekeeping genes with known concentrations in two channels are often used for calibrating experimental parameters. They provide valuable information about experimental variation which can be utilized for better normalization. They are also needed for proper normalization in cases that the most of the spots tend to change in one direction. In addition, it is desirable to include information on spot quality. Such information is available in a typical microarray data set, but is not fully utilized by existing normalization methods. Results: We propose two extensions of the two-way semi-linear model (TW-SLM) for appropriately combining control genes and spot quality information in normalization. The first extension (TW-SLMC) is designed to systematically incorporate control spots in a semi-parametric model to calibrate estimated normalization curves so that the relative fold changes of gene expressions are accurately estimated. Extrapolation is not required in this approach. The second extension (TW-SLMQ) is proposed to incorporate spot quality measure into normalization. This approach down-weights spots with lower quality scores in normalization. These two extensions can be used simultaneously for normalizing a data set. Two microarray data sets are used to demonstrate the proposed methods. Availability: An R based computing package is developed for the proposed methods and available from the corresponding authors. Contact: Deli Wang: deliwang@uab.edu or Jian Huang: jian-huang@uiowa.edu.

Normalization of boutique two-color microarrays with a high proportion of differentially expressed probes

A new normalization method is described for use in specialized boutique arrays which contain a subset of genes selected to test particular biological functions.

Changes in placental adipocytokine gene expression associated with gestational diabetes mellitus

Leptin and adiponectin, two adipocytokines, may work together in regulating energy homeostasis and insulin action. Leptin gene expression has been investigated in term placental tissue complicated by gestational diabetes mellitus (GDM), but never in conjunction with all isoforms of the leptin receptor (LEPR A-D), or with adiponectin receptors (ADIPOR1 and 2). In this study we examined the association between changes in expression of these genes in placental tissue and GDM risk. We assessed placental gene expression of leptin, LEPR A-D and ADIPOR1 and 2 by real time PCR using mRNA from maternal and fetal biopsies. Tissues were collected from uncomplicated pregnancies (n=28) and those complicated by GDM (n=19). Gene expression was normalized to three endogenous housekeeping genes. Relative gene expression values were reported as fold change between groups. Adiponectin gene expression was out of the sensitive range of our assay. There were increases in leptin mRNA expression in GDM cases compared with controls for maternal-side (p=0.06), and fetal-side (p=0.09) placental biopsies. No significant changes were seen in GDM cases compared with controls in LEPR A-D or ADIPOR1 and 2. mRNA derived from maternal-side tissue was positively correlated with tissue from the fetal side for all genes studied (all p<0.01). Finally, we noted that absence or presence of GDM was a major factor in leptin mRNA expression after adjusting for maternal age, mode of delivery, parity and smoking status. In conclusion, increases in leptin mRNA expression in term placenta, but not that of its receptors, are associated with the diagnosis of GDM. Changes seen in the ligand, but not the receptor, of the leptin pathway in GDM-complicated pregnancies may also apply to the adiponectin pathway, as the ADIPOR1 and 2 mRNAs do not change with GDM diagnosis.

266.Human endometrial cycle stages can be determined by global gene expression profiling

Endometrium is a dynamic tissue which undergoes cyclic changes each month, under the overall control of oestrogen and progesterone. The aims of this study were to investigate the changing global gene expression profile of human endometrium during the menstrual cycle using microarray technology and to determine the correlation between histopathological evaluation and molecular profile of the samples. Curettings of endometrium were collected from 43 cycling women and immediately snap frozen. The menstrual cycle was divided into seven stages by histological evaluation. Standard two-color cDNA microarrays were performed on the 43 samples against a common reference, using a 10.5 K cDNA glass slide microarray. Expressed genes were identified using a Scanarray 5000 UV laser scanner. Quantarray software was used to quantify the relative gene expression values. Normalisation and visualisation of the gene expression changes were performed using the GeneSpring software package. Hierarchical clustering of all 43 samples was performed, based on the expression profile of 571 genes, which were identified as differentially expressed by parametric ANOVA with Benjamini-Hochberg correction. The 43 samples were sorted into nine groups which all agreed with histopathology by either being in the same group or an adjacent group apart from four samples. For further analysis, the four outliers were removed, one group was excluded due to lack of replicates and two groups were merged to get the final molecular classification of the cycle. The statistical analysis was repeated and 1452 genes were identified as differentially expressed at P d 0.05. The data were also independently analysed by a CSIRO algorithm called GeneRave and the results from both methods were comparable. mRNA expression profiles of the genes TGF a (Hs.170009), NCR3 (Hs.509513) and FUT4 (Hs.390420) were verified using real-time PCR. We have shown for the first time that endometrial cycle stage prediction is possible based on global gene expression profile.