qPCR array - page 2
qPCR array - page 1
qPCR array application papers
Gene expression profiling in the rhesus macaque: experimental design considerations.
Urbanski HF, Noriega NC, Lemos DR, Kohama SG.
Division of Neuroscience, Oregon National Primate Research Center, Beaverton, OR 97006, USA.
Methods. 2009 Sep;49(1):26-31. Epub 2009 May 23.
The development of species-specific gene microarrays has greatly facilitated gene expression profiling in nonhuman primates. However, to obtain accurate and physiologically meaningful data from these microarrays, one needs to consider several factors when designing the studies. This article focuses on effective experimental design while the companion article focuses on methodology and data analysis. Biological cycles have a major influence on gene expression, and at least 10% of the expressed genes are likely to show a 24-h expression pattern. Consequently, the time of day when RNA samples are collected can influence detection of significant changes in gene expression levels. Similarly, when photoperiodic species such as the rhesus macaque are housed outdoors, some of their genes show differential expression according to the time of year. In addition, the sex-steroid environment of humans and many nonhuman primates changes markedly across the menstrual cycle, and so phase of the cycle needs to be considered when studying gene expression in adult females.
Gene expression profiling in the rhesus macaque: methodology, annotation and data interpretation.
Noriega NC, Kohama SG, Urbanski HF.
Division of Neuroscience, Oregon National Primate Research Center, 505 NW 185th Avenue, Beaverton, OR 97006, USA
Methods. 2009 Sep;49(1):42-9. Epub 2009 May 23.
Gene microarray analyses represent potentially effective means for high-throughput gene expression profiling in non-human primates. In the companion article, we emphasize effective experimental design based on the in vivo physiology of the rhesus macaque, whereas this article emphasizes considerations for gene annotation and data interpretation using gene microarray platforms from Affymetrix. Initial annotation of the rhesus genome array was based on Affymetrix human GeneChips. However, annotation revisions improve the precision with which rhesus transcripts are identified. Annotation of the rhesus GeneChip is under continuous revision with large percentages of probesets under multiple annotation systems having undergone multiple reassignments between March 2007 and November 2008. It is also important to consider that quantitation and comparison of gene expression levels across multiple chips requires appropriate normalization. External corroboration of microarray results using PCR-based methodology also requires validation of appropriate internal reference genes for normalizationod expression values. Many tools are now freely available to aid investigators with microarray normalization and selection of internal reference genes to be used for independent corroboration of microarray results.
A strategy for discovery of cancer glyco-biomarkers in serum using newly developed technologies for glycoproteomics.
Narimatsu H, Sawaki H, Kuno A, Kaji H, Ito H, Ikehara Y.
Research Center for Medical Glycoscience, National Institute of Advanced Industrial Science and Technology, Tsukuba, Japan.
FEBS J. 2010 Jan;277(1): 95-105
Detection of cancer at early stages that can be treated through surgery is a difficult task. One methodology for cancer biomarker discovery exploits the fact that glycoproteins produced by cancer cells have altered glycan structures, although the proteins themselves are common, ubiquitous, abundant, and familiar. However, as cancer tissue at the early stage probably constitutes less than 1% of the normal tissue in the relevant organ, only 1% of the relevant glycoproteins in the serum should have altered glycan structures. Here, we describe our strategy to approach the detection of these low-level glycoproteins: (a) a quantitative real-time PCR array for glycogenes to predict the glycan structures of secreted glycoproteins; (b) analysis by lectin microarray to select lectins that distinguish cancer-related glycan structures on secreted glycoproteins; and (c) an isotope-coded glycosylation site-specific tagging high-throughput method to identify carrier proteins with the specific lectin epitope. Using this strategy, we have identified many glycoproteins containing glycan structures that are altered in cancer cells. These candidate glycoproteins were immunoprecipitated from serum using commercially available antibodies, and their glycan alteration was examined by a lectin microarray. Finally, they were analyzed by multistage tandem MS.
Comparison of breast cancer to healthy control tissue discovers novel markers with potential for prognosis and early detection.
Schummer M, Green A, Beatty JD, Karlan BY, Karlan S, Gross J, Thornton S, McIntosh M, Urban N.
Molecular Diagnostics Program, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America.
PLoS One. 2010 Feb 9;5(2):e9122.
This study was initiated to identify biomarkers with potential value for the early detection of poor-outcome breast cancer. Two sets of well-characterized tissues were utilized: one from breast cancer patients with favorable vs. poor outcome and the other from healthy women undergoing reduction mammaplasty. Over 46 differentially expressed genes were identified from a large list of potential targets by a) mining publicly available expression data (identifying 134 genes for quantitative PCR) and b) utilizing a commercial PCR array. Three genes show elevated expression in cancers with poor outcome and low expression in all other tissues, warranting further investigation as potential blood markers for early detection of cancers with poor outcome. Twelve genes showed lower expression in cancers with poor outcome than in cancers with favorable outcome but no differential expression between aggressive cancers and most healthy controls. These genes are more likely to be useful as prognostic tissue markers than as serum markers for early detection of aggressive disease. As a secondary finding was that, when histologically normal breast tissue was removed from a distant site in a breast with cancer, 7 of 38 specimens displayed a cancer-like expression profile, while the remaining 31 were genetically similar to the reduction mammaplasty control group. This finding suggests that some regions of ipsilateral histologically 'normal' breast tissue are predisposed to becoming malignant and that normal-appearing tissue with malignant signature might warrant treatment to prevent new primary tumors.
MicroRNA expression in ileal carcinoid tumors: downregulation of microRNA-133a with tumor progression.
Ruebel K, Leontovich AA, Stilling GA, Zhang S, Righi A, Jin L, Lloyd RV.
Division of Anatomic Pathology, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN 55905, USA.
Mod Pathol. 2010 Mar;23(3): 367-375
MicroRNAs (miRNAs) are involved in cell proliferation, differentiation, and apoptosis and can function as tumor suppressor genes or oncogenes. The role of miRNAs in neuroendocrine tumors such as ileal carcinoids is largely unknown. We examined the differential expression of 95 miRNAs by RT-PCR using the QuantiMir System in eight matching primary and metastatic carcinoid tumors from the ileum. All miRNAs chosen for the QuantiMir System array were based on their potential functions related to cancer biology, cell development, and apoptosis. The expression of miRNAs for the samples was normalized to miRNA-197, and the matching primary and metastatic tumors were compared. There was downregulation of miRNA-133a, -145, -146, -222, and -10b in all samples between the primary and matching metastatic tumors and upregulation of miRNA-183, -488, and -19a+b in six of eight metastatic carcinoids compared to the primary tumors. miRNA-133a was further analyzed by TaqMan real-time RT-PCR and northern hybridization using six additional matching primary and metastatic samples, which supported the PCR array findings. There were significant differences in miRNA-133a expression with downregulation in the metastasis compared to the primary in the eight original cases (P<0.009) and in the six additional cases used for validation (P<0.014). Laser capture microdissection and real-time RT-PCR analysis using normal ileum found miRNA-133a expression in normal enterochromaffin cells. In situ hybridization in normal ileum showed that some of the mucosal endocrine cells expressed miRNA-133a. Both primary and metastatic ileal carcinoid tumors expressed miRNA-133a by in situ hybridization. These results provide information about novel marker miRNAs that may be used as biomarkers and/or therapeutic targets in intestinal carcinoid tumors.
A Novel Effect of Growth Hormone on Macrophage Modulates Macrophage-Dependent Adipocyte Differentiation.
Lu C, Kumar PA, Fan Y, Sperling MA, Menon RK.
Departments of Pediatrics and Communicable Diseases (C.L., P.A.K., R.K.M.) and Molecular and Integrative Physiology (R.K.M.), University of Michigan, Ann Arbor, Michigan 48109-0718; and Department of Pediatrics (Y.F., M.A.S.), University of
Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15260.
Endocrinology. 2010 Feb 25.
The GH receptor (GHR) is expressed on macrophages. However, the precise role of GH in regulation of macrophage function is unclear. We hypothesized that soluble factors including cytokines produced by macrophages in a GH-dependent manner regulate adipogenesis. We confirmed expression and functional integrity of the GHR in the J774A.1 macrophage cells. Conditioned medium (CM) from macrophages inhibited adipogenesis in a 3T3-L1 adipogenesis assay. CM from GH-treated macrophages decreased the inhibitory effect of CM from macrophages on adipogenesis. This effect on preadipocyte differentiation was active only during the first (early) phase of adipocyte differentiation. CM from stromal vascular compartment macrophages of mice with macrophage-specific deletion of the GHR exhibited more inhibitory effect on 3T3-L1 preadipocyte differentiation compared with CM from stromal vascular compartment macrophages of control mice, indicating that intact GH action in primary macrophages also increases preadipocyte differentiation. GH did not increase IGF-1 expression in macrophages. PCR array analysis identified IL-1beta as a candidate cytokine whose expression was altered by GH in macrophages. Levels of IL-1beta mRNA and protein were significantly decreased in GH-treated J774A.1 macrophages. Nuclear factor-kappaB stimulates IL-1beta gene expression, and GH induced a significant decrease in the levels of phosphorylated nuclear factor-kappaB in macrophages. IL-1beta is a known inhibitor of adipogenesis, and these results support GH-dependent down-regulation of macrophage IL-1beta expression as one mechanism for the observed increase in adipogenesis with CM from GH-treated macrophages. We conclude that GH decreases secretion of IL-1beta by the macrophage and thus in a paracrine manner increases adipocyte differentiation. These results provide a novel mechanism for GH's actions in the control of adipogenesis.
Matrine induces programmed cell death and regulates expression of relevant genes based on PCR array analysis in C6 glioma cells.
Zhang S, Qi J, Sun L, Cheng B, Pan S, Zhou M, Sun X.
Department of Pathology, The First Clinical Medical School of Harbin Medical University, Harbin 150012, China.
Mol Biol Rep. 2009 Apr;36(4): 791-799
Matrine, one of the main components extracted from Sophora flavescens Ait, has a wide range of pharmacological effects including anti-tumor activities on a number of cancer cell lines. This study has investigated whether matrine could also display anti-tumor action on rat C6 glioma cells. Exposure of C6 cells to matrine resulted in inhibition of proliferation and induction of apoptosis in a dose-dependent manner, as measured by the MTT assay and Flow cytometry. The Annexin V/PI staining further detected the apoptotic cells at both early and late phases of apoptosis. We used AO/EB staining to examine the programmed cell death of matrine-treated C6 cells, and showed that the death rate detected by AO/EB staining was higher than the apoptosis rate measured by Annexin V/PI staining, suggesting that autophagy, the Type II programmed cell death, may be involved in matrine-induced cell death, which was further confirmed by electronic microscopy.To explore the molecular mechanism, an apoptosis real-time PCR array was performed, which has demonstrated that 57 genes were at least 2-fold upregulated, and 11 genes were at least 2-fold downregulated in matrine-treated C6 cells, compared with untreated cells. However, the gene expression profiles could only partly and roughly explain molecular mechanisms of apoptosis and autophagy in matrine-treated C6 cells, thus further investigations are required to confirm the specific molecular pathways and related molecules responsible for the programmedcell death.
Spatial expression profiles in the Xenopus laevis oocytes measured with qPCR tomography.
Sindelka R, Sidova M, Svec D, Kubista M.
Whitehead Institute, Cambridge, USA.
Methods. 2010 Jan 4.
qPCR tomography was developed to study mRNA localization in complex biological samples that are embedded and cryo-sectioned. After total RNA extraction and reverse transcription, the spatial profiles of mRNAs and other functional RNAs were determined by qPCR. The Xenopus laevis oocyte was selected as model, because of its large size (more than 1mm) and large amount of total RNA ( approximately 5mug). Fifteen sections along the animal-vegetal axis were cut and prepared for quantification of 31 RNA targets using the high-throughput real-time RT-PCR(qPCR) BioMark platform. mRNAs were found to have two localization patterns, animal/central or vegetal. Because of the high resolution in sectioning, it was possible to distinguish two subgroups of the vegetal gene patterns: germ plasm determinant pattern and profile of other vegetal genes.
LPS induces KH-type splicing regulatory protein-dependent processing of microRNA-155 precursors in macrophages
Tina Ruggiero, Michele Trabucchi,1, Francesca De Santa, Simona Zupo, Brian D. Harfe, Michael T. McManus, M. Geoff Rosenfeld, Paola Briata and Roberto Gherzi
FASEB Vol. 23, 2009
The importance of post-transcriptional mechanisms for the regulation of the homoeostasis of the immune system and the response to challenge by microorganisms is becoming increasingly appreciated. We investigated the contribution of microRNAs (miRNAs) to macrophage activation induced by lipopolysaccharide (LPS). We first observed that Dicer knockout in bone marrow-derived macrophages (BMDMs) increases the LPS-induced expression of some inflammation mediators. miRNA microarray analysis in BMDMs revealed that LPS significantly induces the expression of a single miRNA, miR-155, and this induction depends on enhanced miR-155 maturation from its precursors. The single-strand RNA-binding protein KH-type splicing regulatory protein (KSRP) binds to the terminal loop of miR-155 precursors and promotes their maturation. Both inhibition of miR-155 and KSRP knockdown enhance the LPS-induced expression of select inflammation mediators, and the effect of KSRP knockdown is reverted by mature miR-155. Our studies unveil the existence of an LPS-dependent post-transcriptional regulation of miR-155 biogenesis. Once induced, miR-155 finely tunes the expression of select inflammation mediators in response to LPS.
A real-time PCR array for hierarchical identification of Francisella isolates.
Svensson K, Granberg M, Karlsson L, Neubauerova V, Forsman M, Johansson A.
Division of CBRN Defense and Security, Swedish Defense Research Agency, Umeå, Sweden
PLoS One. 2009 Dec 21;4(12):e8360.
A robust, rapid and flexible real-time PCR assay for hierarchical genetic typing of clinical and environmental isolates of Francisella is presented. Typing markers were found by multiple genome and gene comparisons, from which 23 canonical single nucleotide polymorphisms (canSNPs) and 11 canonical insertion-deletion mutations (canINDELs) were selected to provide phylogenetic guidelines for classification from genus to isolate level. The specificity of the developed assay, which uses 68 wells of a 96-well real-time PCR format with a detection limit of 100 pg DNA, was assessed using 62 Francisella isolates of diverse genetic and geographical origins. It was then successfully used for typing 14 F. tularensis subsp. holarctica isolates obtained from tularemia patients in Sweden in 2008 and five more genetically diverse Francisella isolates of global origins. When applied to human ulcer specimens for direct pathogen detection the results were incomplete due to scarcity of DNA, but sufficient markers were identified to detect fine-resolution differences among F. tularensis subsp. holarctica isolates causing infection in the patients. In contrast to other real-time PCR assays for Francisella, which are typically designed for specific detection of a species, subspecies, or strain, this type of assay can be easily tailored to provide appropriate phylogenetic and/or geographical resolution to meet the objectives of the analysis.
Induction of apoptosis in human bladder cancer cells by green tea catechins.
Philips BJ, Coyle CH, Morrisroe SN, Chancellor MB, Yoshimura N.
Department of Urology, University of Pittsburgh School of Medicine, PA 15213, USA.
Biomed Res. 2009 Aug;30(4): 207-215.
Cell culture and animal studies have demonstrated strong chemopreventative effects of green tea and its associated polyphenols in multiple cancers, though the exact mechanisms of action are not well understood. This in vitro study examined the antiproliferative/pro-apoptotic potential of green tea extract (GTE), polyphenon-60 (PP-60), (-)-epicatechin gallate (ECG) and (-)-epigallocatechin-3-gallate (EGCG) in both normal and malignant human bladder cells. Cell growth (proliferation/apoptosis) was measured in UROtsa (normal), SW780 (tumorigenic; low-grade), and TCCSUP (tumorigenic; high-grade) human bladder urothelial cells by cell proliferation (XTT) assay after treatment with 0-80 microg/mL of GTE, PP-60, ECG and EGCG for 72 h. Molecular signaling pathways of catechin-induced apoptosis were analyzed using Human signal transduction RT(2) Profiler PCR array (SuperArray). Compared to control-treated cells, treatment with catechin agents significantly suppressed cell growth in a dose-dependent fashion (P < 0.01), with strongest effects evoked by ECG and EGCG in UROtsa cells, ECG in low-grade RT4 and SW780 cells, and PP-60 and EGCG in high-grade TCCSUP and T24 cells. Microarray analysis indicated distinct differences in mRNA gene expression regarding growth signaling pathway activation induced by EGCG in normal/tumorigenic human bladder cell lines, providing a rationale for the putative therapeutic usage of green tea polyphenols against bladder disease.
DNA methylation analysis on a droplet-in-oil PCR array.
Zhang Y, Bailey V, Puleo CM, Easwaran H, Griffiths E, Herman JG, Baylin SB, Wang TH.
Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 21218, USA.
Lab Chip. 2009 Apr 21;9(8): 1059-1064
We performed on-chip DNA methylation analysis using methylation-specific PCR (MSP) within an arrayed micro droplet-in-oil platform that is designed for more practical application of microfluidic droplet technologies in clinical applications. Unique features of this ready-to-use device include arrayed primers that are pre-deposited into open micro-reaction chambers and use of the oil phase as a companion fluid for both sample actuation and compartmentalization. These technical advantages allow for infusion of minute amounts of sample for arrayed MSP analysis, without the added complexities inherent in microfluidic droplet-based studies. Ease of use of this micro device is exemplified by analysis of two tumor suppressor promoters, p15 and TMS1 using an on-chip methylation assay. These results were consistent with standard MSP protocols, yet the simplicity of the droplet-in-oil microfluidic PCR platform provides an easy and efficient tool for DNA methylation analysis in a large-scale arrayed manner.
Link to more Lab on Chip publications => Lab-on-Chip PCR & qPCR
Regulation of B Cell Differentiation and Plasma Cell Generation by IL-21, a Novel Inducer of Blimp-1 and Bcl-61
Katsutoshi Ozaki, Rosanne Spolski, Rachel Ettinger, Hyoung-Pyo Kim, Gang Wang, Chen-Feng Qi, Patrick Hwu, Daniel J. Shaffer, Shreeram Akilesh, Derry C. Roopenian, Herbert C. Morse, Peter E. Lipsky, and Warren J. Leonard
Journal of Immunology2004 173(9): 5361-5371.
IL-21 is a type I cytokine whose receptor is expressed on T, B, and NK cells. Within the B cell lineage, IL-21 regulates IgG1 production and cooperates with IL-4 for the production of multiple Ab classes in vivo. Using IL-21-transgenic mice and hydrodynamics-based gene delivery of IL-21 plasmid DNA into wild-type mice as well as in vitro studies, we demonstrate that although IL-21 induces death of resting B cells, it promotes differentiation of B cells into postswitch and plasma cells. Thus, IL-21 differentially influences B cell fate depending on the signaling context, explaining how IL-21 can be proapoptotic for B cells in vitro yet critical for Ag-specific Ig production in vivo. Moreover, we demonstrate that IL-21 unexpectedly induces expression of both Blimp-1 and Bcl-6, indicating mechanisms as to how IL-21 can serve as a complex regulator of B cell maturation and terminal differentiation. Finally, BXSB-Yaa mice, which develop a systemic lupus erythematosus-like disease, have greatly elevated IL-21, suggesting a role for IL-21 in the development of autoimmune disease.
Quantitative Gene Expression Profiling Implicates Genes for Susceptibility and Resistance to Alveolar Bone Loss
G. T. Hart, D. J. Shaffer, S. Akilesh, A. C. Brown, L. Moran, D. C. Roopenian, and P. J. Baker
Infect Immun. 2004 72(8): 4471-4479
Periodontal disease is one of the most prevalent chronic inflammatory diseases. There is a genetic component to susceptibility and resistance to this disease. Using a mouse model, we investigated the progression of alveolar bone loss by gene expression profiling of susceptible and resistant mouse strains (BALB/cByJ and A/J, respectively). We employed a novel and sensitive quantitative real-time PCR method to compare basal RNA transcription of a 48-gene set in the gingiva and the spleen and the subsequent changes in gene expression due to Porphyromonas gingivalis oral infection. Basal expression of interleukin-1 beta (Il1b) and tumor necrosis factor alpha (Tnf) mRNA was higher in the gingiva of the susceptible BALB/cByJ mice than in the gingiva of resistant A/J mice. Gingival Il1b gene expression increased further and Stat6 gene expression was turned on after P. gingivalis infection in BALB/cByJ mice but not in A/J mice. The basal expression of interleukin-15 (Il15) in the gingiva and the basal expression of p-selectin (Selp) in the spleen were higher in the resistant A/J mice than in the susceptible BALB/cByJ mice. In the resistant A/J mice the expression of no genes detectably changed in the gingiva after infection. These results suggest a molecular phenotype in which discrete sets of differentially expressed genes are associated with genetically determined susceptibility (Il1b, Tnf, and Stat6) or resistance (Il15 and Selp) to alveolar bone loss, providing insight into the genetic etiology of this complex disease.
Using real time RT-PCR analysis to determine multiple gene expression patterns during XX and XY mouse fetal gonad development
Gerrit J. Bouma, Geoffrey T. Hart, Linda L. Washburn, Andrew K. Recknagel, Eva M. Eicher
Gene Expr Patterns. 2004 5(1): 141-149
New techniques are being applied to identify all the genes involved in mammalian gonad development and differentiation. As this list of genes increases, understanding the potential interactions between these genes will become increasingly difficult. We used a real time reverse transcription PCR (real time RTPCR) protocol to examine and compare the relative expression levels of 55 genes in individual mouse fetal gonads. Real time PCR analysis demonstrated that except for Sry, no differences in relative gene expression were detectable between XX and XY gonad/mesonephroi complexes at embryonic day (E)11.5. Following Sry peak expression at E11.5, a number of genes were expressed at significantly higher relative levels in E12-14 XY than XX gonads. Of six genes expressed at higher levels in E12.5-14 XX than XY gonads, three, Bmp2, Emx2, and Fgfr2, had not been reported previously. Our results caution that differential localization patterns observed with whole mount in situ hybridization techniques may not accurately reflect changes in transcript levels. We conclude that real time PCR is an efficient and powerful tool for studying multiple gene expression patterns during gonad development and differentiation, and can provide insight into gene interactions.
Congenic Mice Provide in Vivo Evidence for a Genetic Locus that Modulates Serum Insulin-Like Growth Factor-I and Bone Acquisition
K. M. Delahunty, K. L. Shultz, G. A. Gronowicz, B. Koczon-Jaremko, M. L. Adamo, L. G. Horton, J. Lorenzo, L. R. Donahue, C. Ackert-Bicknell, B. E. Kream, W. G. Beamer, and C. J. Rosen
Endocrinology. 2006 147(8): 3915-3923
We identified quantitative trait loci (QTL) that determined the genetic variance in serum IGF-I through genome-wide scanning of mice derived from C57BL/6J(B6) x C3H/HeJ(C3H) intercrosses. One QTL (Igf1s2), on mouse chromosome 10 (Chr10), produces a 15% increase in serum IGF-I in B6C3 F2 mice carrying c3 alleles at that position. We constructed a congenic mouse, B6.C3H-10 (10T), by backcrossing c3 alleles from this 57-Mb region into B6 for 10 generations. 10T mice have higher serum and skeletal IGF-I, greater trabecular bone volume fraction, more trabeculae, and a higher number of osteoclasts at 16 wk, compared with B6 (P < 0.05). Nested congenic sublines generated from further backcrossing of 10T allowed for recombination and produced four smaller sublines with significantly increased serum IGF-I at 16 wk (i.e. 10-4, 10-7, 10-10, and 10-13), compared with B6 (P < 0.0003), and three smaller sublines that showed no differences in IGF-I vs. age- and gender-matched B6 mice. Like 10T, the 10-4 nested sublines at 16 wk had higher femoral mineral (P < 0.0001) and greater trabecular connectivity density with significantly more trabeculae than B6 (P < 0.01). Thus, by comprehensive phenotyping, we were able to narrow the QTL to an 18.3-Mb region containing approximately 148 genes, including Igf1 and Elk-3(ETS domain protein). Allelic differences in the Igf1s2 QTL produce a phenotype characterized by increased serum IGF-I and greater peak bone density. Congenic mice establish proof of concept of shared genetic determinants for both circulating IGF-I and bone acquisition.
The MHC class I – like Fc receptor promotes humorally mediated autoimmune disease
Shreeram Akilesh, Stefka Petkova, Thomas J. Sproule, Daniel J. Shaffer, Gregory J. Christianson, and Derry Roopenian
J Clin Invest. 2004 113(9): 1328-1333
The MHC class I family-like Fc receptor, FcRn, is normally responsible for extending the life span of serum IgG Ab's, but whether this molecule contributes to autoimmune pathogenesis remains speculative. To determine directly whether this function contributes to humoral autoimmune disease, we examined whether a deficiency in the FcRn heavy chain influences autoimmune arthritis in the K/BxN mouse model. FcRn deficiency conferred either partial or complete protection in the arthritogenic serum transfer and the more aggressive genetically determined K/BxN autoimmune arthritis models. The protective effects of an FcRn deficiency could be overridden with excessive amounts of pathogenic IgG Ab's. The therapeutic saturation of FcRn by high-dose intravenous IgG (IVIg) also ameliorated arthritis, directly implicating FcRn blockade as a significant mechanism of IVIg's anti-inflammatory action. The results suggest that FcRn is a potential therapeutic target that links the initiation and effector phases of humoral autoimmune disease.
Real-time PCR array as a universal platform for the detection of genetically modified crops and its application in identifying unapproved genetically modified crops in Japan.
Mano J, Shigemitsu N, Futo S, Akiyama H, Teshima R, Hino A, Furui S, Kitta K.
National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki, Japan.
J Agric Food Chem. 2009 Jan 14;57(1): 26-37
We developed a novel type of real-time polymerase chain reaction (PCR) array with TaqMan chemistry as a platform for the comprehensive and semiquantitative detection of genetically modified (GM) crops. Thirty primer-probe sets for the specific detection of GM lines, recombinant DNA (r-DNA) segments, endogenous reference genes, and donor organisms were synthesized, and a 96-well PCR plate was prepared with a different primer-probe in each well as the real-time PCR array. The specificity and sensitivity of the array were evaluated. A comparative analysis with the data and publicly available information on GM crops approved in Japan allowed us to assume the possibility of unapproved GM crop contamination. Furthermore, we designed a Microsoft Excel spreadsheet application, Unapproved GMO Checker version 2.01, which helps process all the data of real-time PCR arrays for the easy assumption of unapproved GM crop contamination.
The spreadsheet is available free of charge at http://cse.naro.affrc.go.jp/jmano/index.html
Validating nutrient-related gene expression changes from microarrays using RT(2) PCR-arrays.
Gaj S, Eijssen L, Mensink RP, Evelo CT.
Nutrigenomics Consortium, Top Institute Food and Nutrition, Wageningen, The Netherlands
Genes Nutr. 2008 Dec;3(3-4): 153-157
Microarray technology allows us to perform high-throughput screening of changes in gene expression. The outcome of microarray experiments largely depends on the applied analysis methods and cut-off values chosen. Results are often required to be verified using a more sensitive detection technique, such as quantitative real-time PCR (qPCR or RT-PCR). Throughout the years, this technique has become a de facto golden standard. Individual qPCRs are time-consuming, but the technology to perform high-throughput qPCR reactions has become available through PCR-arrays that allow up to 384 PCR reactions simultaneously. Our current aim was to investigate the usability of a RT(2) Profilertrade mark PCR-array as validation in a nutritional intervention study, where the measured changes in gene expression were low. For some differentially expressed genes, the PCR-array confirmed the microarray prediction, though not for all. Furthermore, the PCR-array allowed picking up the expression of genes that were not measurable on the microarray platform but also vice versa. We conclude that both techniques have their own (dis)advantages and specificities, and for less pronounced changes using both technologies may be useful as complementation rather than validation.
Activation of Toll-like receptors 2, 3, and 4 on human melanoma cells induces inflammatory factors.
Goto Y, Arigami T, Kitago M, Nguyen SL, Narita N, Ferrone S, Morton DL, Irie RF, Hoon DS.
Department of Molecular Oncology, John Wayne Cancer Institute, 2200 Santa Monica Boulevard, Santa Monica, CA 90404, USA.
Mol Cancer Ther. 2008 7(11): 3642-3653
Toll-like receptors (TLR) have been shown to be expressed on various types of cancers; however, their functional activity is not known. We examined TLR profiles of human melanoma cells and showed that TLR2, TLR3, and TLR4 were found to be highly expressed. By PCR array analysis, specific stimulation of TLR2, TLR3, and TLR4 on melanoma cells showed significant activation of the adaptor protein MyD88, as well as downstream signal transduction factors nuclear factor-kappaB and inflammatory response-related factors. Specific ligand activation of TLR2, TLR3, and TLR4 was shown to induce cell migration. Peripheral blood lymphocytes and melanoma purified RNA was shown to activate TLR3 on melanoma cells. These studies show expression and functional activity of specific TLRs on melanoma cells and as potential therapeutic targets to control tumor progression.
Gene expression profiling in autoimmune noninfectious uveitis disease.
Li Z, Liu B, Maminishkis A, Mahesh SP, Yeh S, Lew J, Lim WK, Sen HN, Clarke G, Buggage R, Miller SS, Nussenblatt RB.
Laboratory of Immunology, National Eye Institute, National Institutes of Health, Bethesda, MD 20892, USA
J Immunol. 2008 Oct 1;181(7):5147-57.
Noninfectious uveitis is a predominantly T cell-mediated autoimmune, intraocular inflammatory disease. To characterize the gene expression profile from patients with noninfectious uveitis, PBMCs were isolated from 50 patients with clinically characterized noninfectious uveitis syndrome. A pathway-specific cDNA microarray was used for gene expression profiling and real-time PCR array for further confirmation. Sixty-seven inflammation- and autoimmune-associated genes were found differentially expressed in uveitis patients, with 28 of those genes being validated by real-time PCR. Several genes previously unknown for autoimmune uveitis, including IL-22, IL-19, IL-20, and IL-25/IL-17E, were found to be highly expressed among uveitis patients compared with the normal subjects with IL-22 expression highly variable among the patients. Furthermore, we show that IL-22 can affect primary human retinal pigment epithelial cells by decreasing total tissue resistance and inducing apoptosis possibly by decreasing phospho-Bad level. In addition, the microarray data identified a possible uveitis-associated gene expression pattern, showed distinct gene expression profiles in patients during periods of clinical activity and quiescence, and demonstrated similar expression patterns in related patients with similar clinical phenotypes. Our data provide the first evidence that a subset of IL-10 family genes are implicated in noninfectious uveitis and that IL-22 can affect human retinal pigment epithelial cells. The results may facilitate further understanding of the molecular mechanisms of autoimmune uveitis and other autoimmune originated inflammatory diseases.
RNA expression microarrays (REMs), a high-throughput method to measure differences in gene expression in diverse biological samples.
Rogler CE, Tchaikovskaya T, Norel R, Massimi A, Plescia C, Rubashevsky E, Siebert P, Rogler LE.
Department of Medicine and Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, NY, USA. Nucleic Acids Res. 2004 Aug 25;32(15): e120
We have developed RNA expression microarrays (REMs), in which each spot on a glass support is composed of a population of cDNAs synthesized from a cell or tissue sample. We used simultaneous hybridization with test and reference (housekeeping) genes to calculate an expression ratio based on normalization with the endogenous reference gene. A test REM containing artificial mixtures of liver cDNA and dilutions of the bacterial LysA gene cDNA demonstrated the feasibility of detecting transcripts at a sensitivity of four copies of LysA mRNA per liver cell equivalent. Furthermore, LysA cDNA detection varied linearly across a standard curve that matched the sensitivity of quantitative real-time PCR. In REMs with real samples, we detected organ-specific expression of albumin, Hnf-4 and Igfbp-1, in a set of mouse organ cDNA populations and c-Myc expression in tumor samples in paired tumor/normal tissue cDNA samples. REMs extend the use of classic microarrays in that a single REM can contain cDNAs from hundreds to thousands of cell or tissue samples each representing a specific physiological or pathophysiological state. REMs will extend the analysis of valuable samples by providing a common broad based platform for their analysis and will promote research aimed at defining gene functions, by broadening our understanding of their expression patterns in health and disease.