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 programmed
cell 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.
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