Isolation Of mRNA Research Articles

Direct Saliva Transcriptome Analysis

Current standard operating procedures for salivary transcriptomic analysis require low temperatures and lengthy mRNA isolation, which substantially hamper its use in the clinic. We developed a streamlined, ambient-temperature processing, stabilization, and storage protocol for clinical analysis of salivary RNA. The direct saliva transcriptome analysis (DSTA) used cell-free saliva supernatant instead of isolated mRNA for saliva transcriptomic detection, and all procedures, including processing, stabilization, and storage of saliva samples, were performed at ambient temperature without a stabilizing reagent. We evaluated this streamlined protocol by comparing the mRNA expression levels of 3 saliva internal reference genes [glyceraldehyde-3-phosphate dehydrogenase (GAPDH); actin, beta (ACTB); and ribosomal protein S9 (RPS9)] to levels measured with standard procedures, and detecting the variation of their expression levels under long-term ambient temperature storage. The clinical utility of DSTA was assessed by use of 7 oral cancer salivary mRNA biomarkers in a clinical study. Each saliva internal reference gene mRNA showed similar expression levels when assayed by the DSTA or standard procedures, and remained stable under ambient temperature storage for at least 10 weeks without significant degradation (P = 0.918, 0.288, and 0.242 for GAPDH, ACTB, and RPS9, respectively). Compared with standard procedures, the performance characteristics of oral cancer salivary transcriptomic markers were retained as assayed by DSTA after 10 weeks of storage at ambient temperature. These results indicate that the DSTA is a suitable alternative method for saliva transcriptomic analysis and is feasible for use in clinical cancer research applications. The streamlined DSTA protocol can impact the saliva-handling method and improve the standard operating procedures for clinical saliva transcriptomic diagnostics.

The endometrial stromal decidualization miRNA signature

Endometrial stromal decidualization is required for the control of embryonic invasion during implantation, in fact defective decidualization may predispose to pregnancy complications such as preeclampsia, fetal growth restriction or miscarriage. Scarce information is available of how post-transcriprional regulators such us miRNAs control this process. Our objective is to analyze the expression and function of miRNAs during the human decidualization process. Human endometrial stromal cells (hESCs) were decidualized in vitro by estradiol (E2) and progesterone (P4) and the whole genome miRNA expression was analyzed during the decidualization process. hESCs (n = 3) were isolated from endometrial biopsies and then treated with E2 and P4 or vehicle for 9 days and the decidualization status was confirmed by the levels of secreted prolactin and IGFBP-1. Also natural decidualized endometria (n = 5) was obtained and the stromal compartment isolated. After total mRNA isolation and cDNA synthesis, we have investigated the expression of 704 miRNAs using the whole genome miRNA PCR array. T-Student test was used for statistical analysis. We have identified the miRNA decidualization signature using wide genome analysis in a standardized in vitro hESC decidualization model and validated in the in vivo human model. Several miRNAs included in this signature has been reported to be dysregulated in placentas from patients with preeclampsia. Specifically, miR-135b and miR-96 were selected because two of their top predicted gene targets are the transcriptional factors Hoxa10 and Foxo1, genes required for decidualization. miR-135b and miR-96 expression levels decrease significantly in in vitro and in vivo (late proliferative vs late secretory) decidualized stromal cells. hESCs have a specific miRNAs signature that is modified during the decidualization process. These results will be useful for the development of biomarkers of decidualization associated disorders such as preeclampsia.

Rapid induction of interleukin 8 (IL8) expression during prostaglandin F2alpha induced regression of the corpus luteum: role in neutrophil degranulation and chemotaxis

The corpus luteum (CL) is essential for the maintenance of early pregnancy. Prostaglandin F2α (PGF) is a luteolytic factor in many species and has been implicated in luteal regression in women. Recent studies suggest that cytokines may mediate the luteolytic action of PGF. Our objective was to identify cytokines induced by PGF in vivo and to determine their effect on specific luteal cell types in vitro. In vivo and in vitro approaches were used to determine the cytokines produced by the CL. Purified luteal cell types (steroidogenic, endothelial and fibroblast cells) and peripheral blood cells were used to identify which cells respond to cytokines. The study was approved by animal and human studies committees. Midcycle (day 10) cows (n = 15) were injected with saline or a luteolytic dose of PGF. Ovaries were removed after 0.5-4 hr and CL processed for protein or flash frozen for mRNA isolation. Gene expression was analyzed using Affymetrix Bovine Microarrays and validation performed by real-time RT-PCR. Steroidogenic cells, endothelial cells and fibroblasts were prepared for analysis of signaling molecules by western blot and progesterone by RIA. Neutrophils from peripheral blood were analyzed for activation. IL8, CXCL2, CCL2, CCL8 were significantly increased within 60 min of PGF treatment. Ten-fold increases in IL8 were apparent with 60 min and 35 fold increases were observed after 4 hr of treatment. Results confirmed previous findings that PGF induced CCL2 expression. IL8 treatment did not alter basal or LH-stimulated progesterone synthesis. IL8 did not activate AKT or ERK signaling pathways in steroidogenic cells, endothelial cells or fibroblasts. However, IL8 rapidly stimulated neutrophil degranulation as shown by cell surface expression of CDIIB. Cytokines are rapidly induced during luteal regression and IL8 stimulates neutrophil degranulation and chemotaxis, suggesting that luteolysis may be a controlled inflammatory response.

Effects of Postmortem Interval and Preservation Method on RNA Isolated from Field-Preserved Avian Tissues

Studies of gene expression and transcriptome profiling often require the isolation of high-quality mRNA. Here, we examined how preservation method and the lag between collection and processing (postmortem interval; PMI) affect RNA quality and yield in field-collected samples. We subjected tissue samples of 11 House Sparrows (Passer domesticus) to two preservation treatments (flash freezing in liquid nitrogen versus treatment with Ambion's RNAlater prior to freezing) and eight PMI treatments (intervals ranging from 0 to 360 min). Mean RNA yields were significantly higher for flash-frozen tissues. Total RNA yield, as measured by a UV-vis spectro- photometer (Nanodrop), was not negatively associated with PMI in either preservation treatment, and we were able to extract RNA of quality (28S:18S fluorescence ratio >0.9) sufficient for most gene-expression applications from specimens that were held at ambient temperature for up to 6 hr postmortem. These results demonstrate that suffi- cient quantities of high-quality RNA can be extracted from avian tissues collected under typical field conditions. Although RNA remains stable for several hours postmortem, other studies have demonstrated that the transcrip- tional profile changes as a function of PMI. To increase the value of tissue samples for RNA studies we recommend that collectors include with a specimen detailed notes on the duration of the PMI as well as other extrinsic (e.g., time of day, temperature) and intrinsic (e.g., age of bird, sex) factors that influence levels of gene expression.

Dual Kinase/Phosphatase Regulating and Anchoring Protein Tribbles: Expression During Acquisition of Meiotic Competence and Meiosis in Mouse Oocytes.

Mammalian oocytes arrested at prophase of meiosis I undergo a growth phase and acquisition of meiotic competence prior to resumption of meiosis and progression to metaphase of meiosis II. Reversible phosphorylation, involving kinases and phosphatases, is involved in both of these intra-oocyte processes. However, current understandings of how numerous reversible phosphorylation events are orchestrated during acquisition of meiotic competence and maturation are significantly lacking. The Tribbles protein was originally described as a cell cycle regulator required for mesendodermal differentiation and oocyte specification during Drosophila embryogenesis. Drosophila Tribbles protein regulates cell proliferation by promoting degradation of CDC25 protein phosphatase. Mammalian somatic cells contain three Tribbles genes (Tribbles 1, 2, and 3) with more diverse functions, such as anchoring proteins that regulate protein kinase B, mitogen-activated protein kinases and CCAAT/enhancer-binding protein alpha. The objectives of this study were to determine if mammalian oocytes expressed Tribbles and compare Tribbles mRNA levels during acquisition of meiotic competence and maturation. Oocytes were collected from CF1 mice at postnatal d10-13 [growing / germinal vesicle-intact (GV-intact) / meiotically incompetent (Incomp)] and d19-21 following gonadotropin stimulation [GV-intact / meiotically competent (Comp)]. Denuded GV-intact oocytes were pooled and used for mRNA isolation or GV-intact/Comp oocytes were placed into culture and matured for 2, 7, and 16hrs to germinal vesicle breakdown (GVBD), metaphase I (MI) and metaphase II (MII), respectively, prior to mRNA isolation. Gene expression for Tribbles 1, 2, 3, and actin at each stage of oocyte development were quantified by real-time PCR, in triplicate per experiment, in three separate experiments. Expression ratios and/or fold changes were tested for significance by a pairwise fixed reallocation randomization test, and p<0.05 was considered significantly different. Mouse oocytes at all stages assessed expressed all three Tribbles genes. As oocytes gained meiotic competence gene expression of Tribbles 1 and 3 did not significantly change. However, during the transition from GV-intact Incomp to Comp Tribbles 2 gene expression significantly increased 15.7 fold; p<0.001. Tribbles 1 mRNA levels also did not significantly change during transitions from GV-intact to GVBD, to MI, or to MII. During the GV-intact to GVBD transition Tribbles 2 and Tribbles 3 mRNA levels significantly decreased (4.4 fold; p<0.01) and increased (5.7 fold; p<0.01), respectively. Finally, while no significant changes of any Tribbles mRNA occurred during the transition through MI, Tribbles 2 mRNA was found to significantly increase (2.1 fold; p<0.01) during the MI to MII transition. In conclusion, this is the first report of mammalian oocytes expressing all three Tribbles, which are known dual kinase/phosphatase regulatory and anchoring proteins. Tribbles 2 gene expression and mRNA levels change most significantly during acquisition of meiotic competence and meiotic maturation. Continued studies are required at the protein and functional levels and will help elucidate regulatory pathways of meiotic competence acquisition, meiotic maturation, and attainment of embryonic developmental competence. (poster)

Use of urinary microvesicles for noninvasive mRNA expression analysis in patients with prostate cancer.

e15033 Background: Microvesicles (including exosomes) are small lipid bilayer vesicles released from all cells into bodily fluids and have been shown to harbor both RNA and DNA from the parent cell from which they were released. Recently, a rapid method to extract high integrity RNA from microvesicles was developed allowing reliable assessment of their mRNA content. This allows us to gain a transcriptional profile of organs such as the prostate without the need for invasive biopsy. Here we use microvesicles to examine the expression pattern of various genes associated with prostate cancer including the fusion marker TMPRSS:ERG previously shown to be expressed in ~50% of all prostate cancer patients. Methods: A voided (without DRE) or catheterized urine sample was collected from patients with known or suspected prostate cancer from Columbia University Medical Center under approved IRB guidelines. Microvesicles were isolated using a rapid in-house filtration based isolation technique. During RNA extraction a DNase step was included to remove contaminating DNA. RNA integrity was assessed using the Agilent Bioanalyzer. Isolated RNA was used to assess the expression of key prostate markers such as PSA, PCA3 and TMPRSS:ERG. Results: RNA with visible rRNA peaks was successfully isolated from all urine samples assessed. Prostate marker transcripts for PSA, PCA3 and TMPRSS:ERG could be readily detected without the need for a DRE. An initial blinded pilot study of 11 patients revealed that 4 patients were shown to express TMPRSS:ERG mRNA and that these patients corresponded to known prostate cancer patients. TMPRSS:ERG was undetectable in controls and patients with benign prostatic hyperplasia or previously removed prostate cancers. Conclusions: The gene expression profile of prostate cancer patients and controls can be elucidated by isolation of microvesicle mRNA in a voided sample without the need for a DRE. This novel diagnostic tool is currently being investigated in a cohort of 100 cancer patients and controls.

Functional Redundancy between Human SHOX and Mouse Shox2 Genes in the Regulation of Sinoatrial Node Formation and Pacemaking Function

The homeodomain transcription factor Shox2 plays a crucial regulatory role in the development of sinoatrial node (SAN) by repressing the expression of Nkx2.5, as demonstrated by failed differentiation of SAN in Shox2 null mice. The SHOX (short stature homeobox) gene family consists of two closely related members, SHOX and SHOX2 in humans, but a SHOX ortholog does not exist in the mouse genome. These two genes exhibit overlapping and distinct expression patterns in many developing organs but whether they share functional redundancy is not known. In this study, we set to investigate possible functional redundancy between SHOX and SHOX2 in vitro and in vivo. We first showed that human SHOX and SHOX2 and mouse Shox2 possess similar transcriptional repressive activities in cell cultures, particularly the repressive effects on the Nkx2.5 promoter activity. We further created an SHOX/Shox2 knock-in mouse line (replacement of Shox2 with SHOX, referred as Shox2(KI/KI)). Mice carrying the hypomorphic Shox2(KI+Neo/KI+Neo) allele exhibit bradycardia and arrhythmia and die a few days after birth. However, mice carrying the Shox2(KI/KI) allele grow to adulthood. Physiological, histological, and molecular analyses demonstrate a fully developed SAN and normal pacemaking function in Shox2(KI/KI) mice. Our results demonstrate a functional redundancy between human SHOX and mouse Shox2 in the regulation of SAN formation and pacemaking function in addition to several other organs. The SHOX/Shox2 dose appears to be critical for normal pacemaking function.

2321 COMPARATIVE ANALYSIS OF PROSTATE CANCER ANTIGEN 3 MRNA EXPRESSION IN BENIGN PERIPHERAL PROSTATIC TISSUE, CANCER AND ISOLATED OR CANCER-ASSOCIATED HIGH GRADE PROSTATIC INTRAEPITHELIAL NEOPLASIA

You have accessJournal of UrologyProstate Cancer: Markers1 Apr 20112321 COMPARATIVE ANALYSIS OF PROSTATE CANCER ANTIGEN 3 MRNA EXPRESSION IN BENIGN PERIPHERAL PROSTATIC TISSUE, CANCER AND ISOLATED OR CANCER-ASSOCIATED HIGH GRADE PROSTATIC INTRAEPITHELIAL NEOPLASIA M. Teresa Quiles, Maria Antònia Arbós, Inés M. de Torres, Carmen Blázquez, Juan M. Bastarós, José Placer, Andreas Doll, Jaume Reventós, and Juan Morote M. Teresa QuilesM. Teresa Quiles Barcelona, Spain More articles by this author , Maria Antònia ArbósMaria Antònia Arbós Barcelona, Spain More articles by this author , Inés M. de TorresInés M. de Torres Barcelona, Spain More articles by this author , Carmen BlázquezCarmen Blázquez Barcelona, Spain More articles by this author , Juan M. BastarósJuan M. Bastarós Barcelona, Spain More articles by this author , José PlacerJosé Placer Barcelona, Spain More articles by this author , Andreas DollAndreas Doll Barcelona, Spain More articles by this author , Jaume ReventósJaume Reventós Barcelona, Spain More articles by this author , and Juan MoroteJuan Morote Barcelona, Spain More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.2568AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Prostate cancer antigen 3 (PCA3) score in urine after prostate-massage is a novel tool to increase prostate specific antigen (PSA) specificity especially in men with repeat prostate biopsy indication. Current data suggest a lower efficacy of PCA3 score in men with HGPIN in the first biopsy1. The objective of this study was to analyze the PCA3 gene expression in benign tissue (BT), cancer (CA) and HGPIN lesions (isolated: IHGPIN and cancer-associated: AHGPIN). METHODS Total mRNA was isolated and amplified from laser-assisted microdissected foci of BT, CA and AHGPIN located in 15 radical prostatectomy frozen specimens and IHGPIN located in 13 radical cistoprostatectomy paraffin-embedded blocks from patients with bladder cancer. PCA3 mRNA amounts were measured by real-time qPCR (Sybr Green I chemistry). PCA3 mRNA amounts were normalized to two reference genes: small nucleolar RNA (SNORD) and Rho GTPase activating protein (ARHGAP). Target gene levels were determined using efficiency-corrected calibrator normalized relative quantification. Comparison among tissues was performed using non-parametric Friedman test followed by post-hoc Dunn's multiple comparison test. RESULTS PCA3 mRNA expression was detected in 80% of BT samples, 86.7% of AHGPIN and 92.9% of CA tissues. PCA3 mean quantitative expression was 11.4 (p=0.009) and 20.7-fold increased (p=0.0002) in AHGPIN and CA related to BT samples. Qualitative PCA3 mRNA expression was detected in 61.5% of IHGPIN samples. CONCLUSIONS We have demonstrated that PCA3 is not a specific gene of prostate cancer. PCA3 mRNA expression is significantly increased in cancer-associated HGPIN lesions as well as qualitatively expressed in isolated HGPIN lesions. After these findings and the previous clinical observation of a lower efficacy of PCA3 score in men with previous HGPIN1 we suggest that specific threshold of PCA3 score should be established in those men. References 1 : Behavior of the PCA3 gene in the urine of men with high grade prostatic intraepithelial neoplasia. World J Urol2010 Jul 7; . [Epub ahead of print]. Google Scholar © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e930-e931 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information M. Teresa Quiles Barcelona, Spain More articles by this author Maria Antònia Arbós Barcelona, Spain More articles by this author Inés M. de Torres Barcelona, Spain More articles by this author Carmen Blázquez Barcelona, Spain More articles by this author Juan M. Bastarós Barcelona, Spain More articles by this author José Placer Barcelona, Spain More articles by this author Andreas Doll Barcelona, Spain More articles by this author Jaume Reventós Barcelona, Spain More articles by this author Juan Morote Barcelona, Spain More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

A clone-based transcriptomics approach for the identification of genes relevant for itaconic acid production in Aspergillus

Several Aspergillus species are well-known for the production of a variety of organic acids. In this study, a cloned based transcriptomics approach was used to identify genes crucial in the biosynthesis pathway for one of these acids, itaconic acid. From a number of different Aspergillus terreus controlled batch fermentations, those cultures with the largest difference in itaconic acid titer and productivity were selected for mRNA isolation. cDNAs derived from these mRNA samples were used for subsequent hybridization of glass slide arrays based on a collection of 5000 cDNA clones.A selection of 13 cDNA clones resulting in the strongest (>10-fold) differential hybridization signals were identified and subsequently the inserts of these clones were sequenced. Sequence analysis revealed the presence of in total five different gene inserts among the sequenced clones. From one of these sequences, encoding a gene of the MmgE–PrpD family, the full length coding region was shown to encode one of the crucial itaconic acid pathway enzymes cis-aconitate decarboxylase, by heterologous expression in Escherichia coli. Expression of this gene in Aspergillus niger, which is a natural citric acid producer, resulted in itaconate production. Genome analysis suggests that in A. terreus the cis-aconitate decarboxylase gene is part of an itaconate acid related gene cluster including genes encoding two pathway specific transporters and a Zinc finger protein. Interestingly, this cluster is directly linked to the large lovastatin gene cluster.

The effect of fenugreek on the gene expression of arachidonic acid metabolizing enzymes

The main bioactive compounds of Trigonella foenum graecum L. (fenugreek) seeds are protodioscin, trigoneoside, diosgenin and yamogenin, which have anticarcinogenic potency through inhibition of cell proliferation and inhibition of prostaglandin synthesis. The effect of fenugreek on ALOX and COX genes was examined in AKR/J H-2(k) mice exposed to dimethylbenz[α]anthracene (DMBA), a potent carcinogen. The expression pattern of these genes was determined by detecting the mRNA expression in various tissues (the lungs, liver, spleen and the kidneys) in four groups of mice. Two groups were fed with normal and two of them with fenugreek containing nutriment. Each group divided into DMBA treated and control groups. Mice were autopsied on day 7 after DMBA treatment for mRNA isolation. Fenugreek consumption itself did not change gene expression compared with the control group. DMBA could increase the expression of ALOX12, ALOX15, ALOX5 genes mainly in all organs. Fenugreek consumption was generally protective in each organ in a different manner. DMBA treatment increased COX2 gene expression, but fenugreek was protective in all tissues examined. In COX1 gene, the fenugreek diet could suppress the expression, except for spleen, independently from carcinogen exposure. Therefore by inhibiting the arachidonic acid metabolism fenugreek may prevent tumorigenesis.

Microfluidic robotic device coupled with electrochemical sensor field for handling of paramagnetic micro-particles as a tool for determination of plant mRNA

The expression of genes responsible for the biosynthesis of stress proteins corresponds to the exposition of an organism to abiotic and/or biotic stress. We utilize two types of paramagnetic particles for isolation of total mRNA from early somatic embryos of Norway Spruce (Picea abies /L./ Karst.) and maize plants (Zea mays L.) treated with cadmium(II) ions. The paramagnetic particles were evaluated for analysis of real samples, and poly-adenine was used as a model mRNA. Various approaches (from non-automatic to fully automatic) were tested in terms of handling the particles.

Anti-CA19-9 Diabody As A PET Imaging Probe For Pancreas Cancer

Introduction: Numerous antibodies have been developed against tumor specific antigens on the cancer cell surface. For this reason, radiolabeled antibodies have the potential to be sensitive and specific PET imaging agents. Intact antibodies, however, are limited by a long serum half-life (10-20days) preventing adequate contrast between the tumor and surrounding blood. Smaller engineered antibody fragments overcome this problem by exhibiting shorter serum half-lives (4-20hrs).the diabody is the smallest antibody fragment which retains the divalency of the intact antibody. It is composed of the variable heavy and light chain and is approximately 55 kDa. Our goal was to develop and characterize the Anti-CA19-9 Diabody fragment and determine its ability to provide sensitive and specific imaging of pancreas cancer. Methods: Expression of CA19-9 was evaluated using flow cytometry on human cancer cell lines (BxPC3, Capan-2, MiaPaca-2). The diabody DNA construct was created by isolation of the variable region mRNA and utilization of RT-PCR. This construct was ligated into the pEE12 vector for transfection into NSO mouse myeloma cells for screening and production of clones that secrete the anti-CA19-9 diabody. The diabody was purified using HPLC and evaluated in vitro with flow cytometry and immunofluorescence to confirm binding ability. The diabody was labeled with a positron emitting radionuclide (Iodine-124) and injected into mice harboring an antigen positive xenograft (BxPC3 or Capan-2) and a negative xenograft (MiaPaca). MicroCT and MicroPET were performed at successive time intervals after injection. Radioactivity was measured in blood and tumor to provide objective confirmation of the microPET images. Results: Immunofluorescence and flow cytometry showed specific binding of the Anti-CA19-9 Diabody comparable to that of the parental intact antibody. Pancreas xenograft imaging of BxPC3/MiaPaca-2 with the Anti-CA19-9 Diabody demonstrated an average tumor:blood ratio of 5.0 and positive:negative tumor ratio of 8.8. Additionally, imaging of pancreatic xenografts, Capan-2/MiaPaca-2, demonstrated an average tumor:blood ratio of 2.04 and positive:negative tumor ratio of 6.2. See Figure 1. Conclusions: Molecular imaging of pancreas cancer should improve our ability to evaluate extent of disease as well as the effect of systemic therapies. To this end, we have created and characterized an Anti-CA19-9 Diabody. Furthermore, we demonstrate that Anti-CA19-9 Diabody fragment PET imaging achieves adequate tumor to blood ratios to provide sensitive and specific molecular imaging of pancreas cancer in tumor xenograft models. Additionally, the diabody fragment has the potential to provide specific tumor targeting of therapeutic nanoparticles.

Oligonucleotide immobilization using 10-(carbomethoxy)decyl-dimethylchlorosilane for mRNA isolation and cDNA synthesis on a microfluidic chip

Fabrication of microfluidic systems capable of extracting and isolating nucleic acids from biological samples and preparing them for downstream applications within the same device is of interest as on-chip sample preparation reduces the time and effort expended on multi-step benchtop procedures. A microfluidic chip capable of cell lysis, nucleic acid extraction and immobilization has been developed in our laboratory. This report focuses on substrate development and chip integration of the nucleic acid immobilization platform. The immobilization region was initially developed using poly-adenylated mRNA released from normal human lymphoblastoid cells. After immobilization, the mRNA remains functional for amplification by the reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time PCR (qPCR) gene expression analyses. Detailed surface functionalization, chemical modification, and covalent oligonucleotide immobilization methods are described. Vapor and liquid deposition techniques for Pyrex functionalization by 10-(carbomethoxy)decyl-dimethylchlorosilane (CMDCS) were developed and an optimal method was determined for microfluidic chip application. CMDCS substrate depositions and modification were characterized by atomic force microscopy and contact angle goniometry, respectively. Oligonucleotide immobilizations and mRNA bindings were determined by fluorescence microscopy. Functional, selective platform integration within the microfluidic chip was confirmed by standard RT-PCR and qPCR systems.

Abstract P1-13-07: Standardized Immunocytological and Molecular Analysis of Tumor Cells and Mesenchymal Stromal Cells in Bone Marrow of Patients with Primary Breast Cancer

Abstract Background: Using criteria for classification of disseminated tumor cells in compliance with the publication of Fehm et al. (Cancer, Vol.107, 2006) we have reported a cytological DTC-positivity rate of 1.9% (39/2050) in patients with primary breast cancer (T1-4, N0/N+, M0/M1). However automated picture analysis revealed cytokeratin positive stroma cell types in DTC negative samples. Here we describe the cytological and molecular characterization of these mesenchymal stromal cell (MSC) types. Material and Methods: Mononuclear cells were enriched by Ficoll density-gradient centrifugation. Cytospin slides (mean 6) with 1x106 BM-cells were stained by CK antibodies 5D3 and A45-B/B3 using automated immunostainers with the APAAP-and the enhanced SA-FastRed-method. Slides were analysed by automated picture analysis with the ACIS I and ACIS II systems (ChromaVision). Plastic adherent cells (1x106) from culture passage one from 71 breast cancer patients were analysed for expression of CK19, MUC1 and EpCAM by FACS analysis. Trypsinized cells (n=100) were lysed and used for mRNA isolation and c-DNA synthesis (Qiagen®). A real-time quantitative RT-PCR approach (MESA FAST SYBR Assay, Eurogentec®) and primers selected from the UniversalProbeLibrary system (Roche AG®) was used for the determination of the markers EpCAM, survivin, CD144, CD146, CD 105, and CD271. Results: Small cells characterized by an excentrically located small and clear nucleus were detected in 56% (486/866) of the bone marrow samples in a mean frequency of 1-2 cells per slide. However a subgroup of patients is characterized by elevated MSC levels. More than 5 MSC cells per 106 BM cells were detected in 19,7% (96/486) samples of the DTC negative group with elevated MSC levels in 5,2% NO, 6% N+ and 9% T3/T4 tumors. By molecular expression analysis of 71 bone marrow samples 8 probes (11%) were CK19 positive. Corresponding MSC cultures had significantly elevated expression levels of the markers vimentin, survivin, CD144, CD105, CD146 and CD271. Discussion: We have identified CK positive bone marrow stroma cells in 56% of patients with primary breast cancer. Immunobead RT-PCR based on 107 BM cells can be used to eliminate this background and to increase DTC-sensitivity from 2 to 11%. Frequency and expression profile of MSC in these probes may give valuable additional information to understand in the complex interrelation of tumor cells with tumor-associated mesenchymal stromal cells, erythroblasts and endothelial cells in the process of metastasis. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P1-13-07.

FoxP3, GATA‐3 and T‐bet expression in elderly asthma

Asthma is a chronic inflammatory disorder in which Th2, Th1 and suppressive T cells (Tregs) play a role. The transcription factor FoxP3 plays a role in Treg differentiation while T-bet is important for Th1 and GATA-3 for Th2 differentiation from naïve T cells. Recent data show that age-related deregulation of Treg cells is a mechanism of senescence affecting several chronic diseases. It is crucial to understand the behaviour of these cell populations in asthma for elderly patients. To evaluate FoxP3, GATA-3 and T-bet gene expression under basal conditions and after in vitro activation in a group of elderly asthmatic compared with age-matched healthy individuals. Thirty-two elderly asthmatics and 17 healthy elderly individuals were selected. Serum total IgE was measured, and peripheral blood mononuclear cells (PBMCs) were isolated and stimulated in vitro with anti-CD3/anti-CD28, followed by mRNA isolation. After reverse transcription, real-time quantitative PCR was performed and relative quantification was determined 2(-ΔΔCt)(2(-ΔΔCt) method). The mean values and standard deviation of FoxP3, GATA-3 and T-bet relative expression for control vs. asthma were 10.2±6.8 vs. 4.8±3.8, 2.4±2.9 vs. 1.7±0.9 and 3.3±2.1 vs. 2.1±1.5, respectively. Healthy individuals showed significantly higher expression of FoxP3 and T-bet; asthmatics had a lower T-bet/GATA-3 ratio, higher serum IgE and a positive significant correlation between total IgE and GATA-3 expression. Elderly asthmatic patients have lower FoxP3 mRNA expression in PBMC, which can be associated with the sustained inflammatory process and with the decreased immune tolerance by Treg cells. The T-bet deficiency and the correlation of GATA-3 expression with the increase of IgE are characteristics of long-lasting asthma. Changes related to the immunosenescence process could provide an explanation for the minor differences observed between the groups. It is important to clarify persistent modifications in long-lasting asthma in the elderly and adequate future therapeutic approaches.

How to Isolate Polysomal RNA to Study Mammalian Early Development.

Pre-hatching development, which encompasses oocyte maturation until blastocyst escape from the zona pellucidae, can be boldly described as two distinct developmental windows, according to the transcriptional potential of the cells. The first window is characterized by transcriptional silence, where cells support protein synthesis by recruiting stored maternal RNAs, whereas in the second interval, the embryonic genome is active and thus capable of producing de novo mRNAs. The presence of large amounts of stored transcripts destined to be either transcribed later on during development or simply sent for decay creates a large background noise that hinders the potential to gain relevant knowledge on the making of pre-hatching development, either through low (real-time RT-PCR) or high throughput (microarray, deep sequencing) methods. We believe that the study of mRNAs in the process of being translated might offer a better perspective of the making of an early embryo. Consequently, we developed a method that enables the isolation of messenger RNAs found to be bound to ribosomes using sucrose gradient fractionation. The main constraint to achieve such an objective is the small amount of starting material. Here, we present the development of a reliable method that enables the survey of the sub-population of mRNAs found in the polysomal fraction using as little as 100 oocytes or embryos. The method utilizes carrier polysomes prepared from a distant species to provide a confirmation of the polysomal nature of the isolated mRNAs. With such an approach, a method is required to eliminate the contribution of carrier RNA on downstream steps. To do so, ribonucleoprotein complexes are cross-linked and the phylogenetic distance between the carrier and the species of interest was proven to almost completely prevent the potential contribution of the carrier on microarray results. The method was tested to confirm its high repeatability by performing a survey of GV stage polysomal mRNAs using microarray hybridization. The mean correlation value between technical replicates was 0.95. We tested the method in a physiological context by comparing the mRNAs found in the polysomal fractions of GV, GVBD and MII stage bovine oocytes. Survey of the identity of the mRNA found in the polysomal fractions was conducted using microarray and quantitative RT-PCR. Translation behaviour of candidates studied by quantitative RT-PCR (C-Mos, Cyclin B1 and CDK1) is correlated with the underlying physiology of oocyte maturation. Polysomal RNA abundance was shown not to correlate with the corresponding protein levels as polysomal synthesis is supplementary to the initial protein content. To our knowledge, this is the first successful isolation of mRNA confirmed to be of polysomal nature and allowing specific candidate studies. Polysomal fractionation provides a novel angle to study early development that may be more insightful than the study of the entire mRNA content. (poster)

Expression Profile of Connexin 43 and Poly A Polymerase Genes in Buffalo (Bubalus bubalis) Oocytes and Developing Embryos Produced in vitro

Mishra, A., Sharma, G.T. and Kumar, G.S. 2010. Expression profile of connexin 43 and poly A polymerase genes in buffalo (Bubalus bubalis) oocytes and developing embryos produced in vitro. J. Appl. Anim. Res., 38: 29–32. To find out the expression pattern of Connexin 43 (Cx43) a gap junction protein and Poly A polymerase (PAP) necessary for synthesis of poly (A) tail in mRNA, genes in buffalo (Bubalus bubalis) oocytes and developing embryos produced in vitro, pools of immatured oocytes (n=200), in vitro matured oocytes (n=200), embryos of 2–4 cell (n=83), 8–16 cell (n=80), morula (n=77) and blastocyst (n=40) were collected in vitro from slaughter house ovaries for mRNA isolation. RT-PCR was carried out by using mRNA as the template and PCR was carried out by using cDNA as template for amplification of 425 bp Cx43 and 252 bp PAP. Cx43 was expressed in all except blastocysts stage, whereas, PAP was expressed in all. It is concluded that expression pattern of Cx43 and PAP in oocytes and developing embryos can be used as a marker of developmental potential for embryos derived from oocytes.

A11 Alterations in the PGC-1 α dependent oxidative stress response in the R6/2 mouse model of HD

BackgroundIn Huntington's disease (HD), alterations in energy metabolism and mitochondrial function may result in oxidative stress. Increased levels of oxidative damage products have been reported in HD. The transcriptional co-activator...

Clinical microfluidics for neutrophil genomics and proteomics

Neutrophils play critical roles in modulating the immune response. We present a robust methodology for rapidly isolating neutrophils directly from whole blood and develop ‘on-chip’ processing for mRNA and protein isolation for genomics and proteomics. We validate this device with an ex vivo stimulation experiment and by comparison with standard bulk isolation methodologies. Lastly, we implement this tool as part of a near patient blood processing system within a multi-center clinical study of the immune response to severe trauma and burn injury. The preliminary results from a small cohort of patients in our study and healthy controls show a unique time-dependent gene expression pattern clearly demonstrating the ability of this tool to discriminate temporal transcriptional events of neutrophils within a clinical setting.

Restoration of Dlk1 and Rtl1 Is Necessary but Insufficient to Rescue Lethality in Intergenic Differentially Methylated Region (IG-DMR)-deficient Mice

In the Dlk1-Dio3 imprinted domain, an intergenic differentially methylated region (IG-DMR) regulates the parental allele-specific expression of imprinted genes. The maternally inherited deletion of IG-DMR (IG-DMR((-/+))) results in perinatal lethality because of the overexpression of paternally expressed genes and repression of maternally expressed noncoding RNAs (ncRNAs), including Gtl2. To better understand the possible contribution of paternally expressed genes to the lethality, we attempted to rescue the lethality of IG-DMR((-/+)) mutants by restoring the paternally expressed genes. Because the paternally inherited Gtl2 deletion (Gtl2((+/-))) induced a decrease in the expression of paternally expressed genes, we crossed female IG-DMR heterozygous mice and male Gtl2 heterozygous mutant mice. The resultant IG-DMR((-/+))/Gtl2((+/-)) double mutant mice had normal expression levels of paternally expressed genes, and none of them showed perinatal lethality; however, most mice showed postnatal lethality with decreased expression of the maternally expressed ncRNAs. Thus, we inferred that paternally expressed genes are necessary for perinatal survivability and that maternally expressed ncRNAs are involved in postnatal lethality.