Development and Validation of a Harmonized TaqMan-Based Triplex Real-Time RT-PCR Protocol for the Quantitative Detection of Normalized Gene Expression Profiles of Seven Porcine Cytokines

Purpose Traditional genetic approaches to identify gene mutations in cancer are expensive and laborious. Nonetheless, if we are to avoid rejecting effective molecular targeted therapies, we must test these drugs in patients whose tumors harbor mutations in the drug target. We hypothesized that gene expression profiling might be a more rapid and cost-effective method of identifying tumors that contain specific genetic abnormalities. Materials and Methods Gene expression profiles of 46 samples of medulloblastoma were generated using the U133av2 Affymetrix oligonucleotide array and validated using real-time reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry. Genetic abnormalities were confirmed using fluorescence in situ hybridization (FISH) and direct sequencing. Results Unsupervised analysis of gene expression profiles partitioned medulloblastomas into five distinct subgroups (subgroups A to E). Gene expression signatures that distinguished these subgroups predicted the presence of key molecular alterations that we subsequently confirmed by gene sequence analysis and FISH. Subgroup-specific abnormalities included mutations in the Wingless (WNT) pathway and deletion of chromosome 6 (subgroup B) and mutations in the Sonic Hedgehog (SHH) pathway (subgroup D). Real-time RT-PCR analysis of gene expression profiles was then used to predict accurately the presence of mutations in the WNT and SHH pathways in a separate group of 31 medulloblastomas. Conclusion Genome-wide expression profiles can partition large tumor cohorts into subgroups that are enriched for specific genetic alterations. This approach may assist ultimately in the selection of patients for future clinical trials of molecular targeted therapies.

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Validation of Array-Based Gene Expression Profiles by Real-Time (Kinetic) RT-PCR

Journal of Molecular Diagnostics ◽

10.1016/s1525-1578(10)60646-0 ◽

2001 ◽

Vol 3 (1) ◽

pp. 26-31 ◽

Cited By ~ 198

Author(s):

Mangalathu S. Rajeevan ◽

Suzanne D. Vernon ◽

Naovarath Taysavang ◽

Elizabeth R. Unger

Keyword(s):

Gene Expression ◽

Real Time ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Rt Pcr

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COMPARISON OF GENE EXPRESSION PROFILES PRODUCED BY CAGE, ILLUMINA MICROARRAY AND REAL TIME RT-PCR

Genome Informatics 2010 ◽

10.1142/9781848166585_0005 ◽

2010 ◽

Author(s):

ANDRÉ FUJITA ◽

MASAO NAGASAKI ◽

SEIYA IMOTO ◽

AYUMU SAITO ◽

EMI IKEDA ◽

...

Keyword(s):

Gene Expression ◽

Real Time ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Rt Pcr

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Quantitative Gene Expression Profiles in Real Time From Expressed Sequence Tag Databases

Gene Expression ◽

10.3727/105221610x12717040569820 ◽

2010 ◽

Vol 14 (6) ◽

pp. 321-336 ◽

Cited By ~ 3

Author(s):

VINCENT A. FUNARI ◽

KONSTANTIN VOEVODSKI ◽

DIMITRY LEYFER ◽

LAURA YERKES ◽

DONALD CRAMER ◽

...

Keyword(s):

Gene Expression ◽

Real Time ◽

Expressed Sequence Tag ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Quantitative Gene Expression ◽

Expressed Sequence

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410. Gene expression analysis in endometriotic lesions using laser capture microdissection

Reproduction Fertility and Development ◽

10.1071/srb08abs410 ◽

2008 ◽

Vol 20 (9) ◽

pp. 90

Author(s):

L. Fu ◽

J. E. Girling ◽

P. A. W. Rogers

Keyword(s):

Gene Expression ◽

Real Time ◽

Laser Capture Microdissection ◽

Stromal Cells ◽

Expression Profiles ◽

Specific Gene ◽

Rt Pcr ◽

Normal Endometrium ◽

Rna Quality ◽

Laser Capture

Previous studies examining gene expression profiles in normal endometrium and endometriotic lesions have used RNA extracted from whole tissue samples. Results from these studies can be difficult to interpret as they reflect expression averaged across several different cell types that may be functionally quite different. The aim of this study was to establish laser capture microdissection (LCM) as a technique to examine gene expression in stromal and epithelial cells from normal and ectopic endometrium. We hypothesised that genes associated with inflammation would be elevated in cells from endometriotic lesions. Full thickness uterine samples were collected during abdominal hysterectomy from normal cycling premenopausal women. Endometriotic lesions were collected during abdominal laparoscopy. Samples were either frozen in OCT or stored in RNAlater for 12 h before freezing. Tissues were immunostained with an antibody against CD10 to identify ectopic endometrial stromal cells before LCM. Endometrial epithelial and stromal cells were collected using the PALM MicroLaser System. RNA quality was accessed using Experion. TGFβ1, MMP1, αSMA, SMAD2 and NFκB mRNA was analysed using real-time RT–PCR. Of the endometriotic samples stored in OCT (n = 58), only 14% (n = 8) had visible endometrial glands. Of these, only 37% (n = 3) had RNA of an acceptable quality for further analysis. However, RNA quality and quantity were dramatically improved in 3 of 5 samples collected in RNAlater. In preliminary studies, expression of TGFβ1 and αSMA mRNA was elevated in endometriotic lesions in comparison to the normal endometrium, whereas NFκB expression did not change. We have shown that RNAlater solution is useful to preserve RNA quality for small clinical endometriotic samples and that immuno-guided LCM-generated homogenous cell populations coupled with real-time RT–PCR can provide valuable insights into cell and disease-specific gene expression in endometriotic lesions.

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Assessment of gene expression profiles in primary and secondary zoospores of Plasmodiophora brassicae by dot blot and real-time PCR

Microbiological Research ◽

10.1016/j.micres.2013.02.011 ◽

2013 ◽

Vol 168 (8) ◽

pp. 518-524 ◽

Cited By ~ 18

Author(s):

Jie Feng ◽

Sheau-Fang Hwang ◽

Stephen E. Strelkov

Keyword(s):

Gene Expression ◽

Real Time ◽

Real Time Pcr ◽

Plasmodiophora Brassicae ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Dot Blot

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MLL-AF9 and MLL-ENL Induce Deregulation of Similar Downstream Candidate Genes: An Analysis across Experimental Protocols and Laboratories.

Blood ◽

10.1182/blood.v104.11.3372.3372 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3372-3372

Author(s):

Ashish R. Kumar ◽

Robert K. Slany ◽

Jay L. Hess ◽

John H. Kersey

Keyword(s):

Gene Expression ◽

Fusion Protein ◽

Fusion Gene ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Expression Systems ◽

Rt Pcr ◽

Conditional Expression

Expression profiling has become an important tool for understanding gene deregulation in MLL-fusion leukemias. However, the results of gene profiling experiments are difficult to interpret when applied to leukemia cells because (i) leukemias arise in cells that differ greatly in their gene expression profiles, and (ii) leukemias most often require secondary genetic events in addition to the MLL fusion gene. Two principal model systems have been used to understand the direct effects of MLL-fusion genes. Knock-in models have the advantage of the fusion gene being under control of the physiologic promoter. On the other hand, conditional expression systems offer the ability to conduct short term experiments, permitting the analysis of direct effects on downstream genes. In the present combined-analysis, we used the Affymetrix U74Av2 oligonucleotide microarray to evaluate the effects of the MLL-fusion gene in vivo and in vitro respectively using two closely related MLL fusion genes - MLL-AF9 for knock-in and MLL-ENL for conditional expression. In the MLL-AF9 study, we compared gene expression profiles of bone marrow cells from MLL-AF9 knock-in mice (C57Bl/6, MLL-AF9+/−) to those of age-matched wild type mice (Kumar et. al. 2004, Blood). We used a t-test (p<0.05) to selected genes that showed significant changes in expression levels. In the MLL-ENL study, we transformed murine primary hematopoietic cells with a conditional MLL-ENL vector (MLL-ENL fused to the modified ligand-binding domain of the estrogen receptor) such that the fusion protein was active only in the presence of tamoxifen. We then studied the downstream effects of the fusion protein by comparing gene expression profiles of the cells in the presence and absence of tamoxifen. We used a pair-wise comparison analysis to select genes that showed a change in expression level of 1.5 fold or greater in at least two of three experiments (Zeisig et. al. 2004, Mol. Cell Biol.). Those genes that were up-regulated in both datasets were then compiled together. This list included Hoxa7, Hoxa9 and Meis1. The results for these 3 genes were confirmed by quantitative RT-PCR in both the MLL-AF9-knock-in and the MLL-ENL-conditional-expression systems. The remaining candidate genes in the common up-regulated gene set (not yet tested by quantitative RT-PCR) include protein kinases (Bmx, Mapk3, Prkcabp, Acvrl1, Cask), RAS-associated proteins (Rab7, Rab3b), signal transduction proteins (Notch1, Eat2, Shd, Fpr1), cell membrane proteins (Igsf4), chaperones (Hsp70.2), transcription factors (Isgf3g), proteins with unknown functions (Olfm1, Flot1), and hypothetical proteins. The results of the combined analysis demonstrate that these over-expressions are (i) a direct and sustained effect of the MLL-fusion protein, (ii) are independent of secondary events that might be involved in leukemogensis, and (iii) are independent of the two partner genes that participate in these fusions. The over-expression of a few genes in both the -in vitro and in vivo experimental systems makes these molecules very interesting for further studies, to understand the biology of MLL-fusion leukemias and for development of new therapeutic strategies.

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Early T-Cell Precursor-ALL in Adult T-ALL.

Blood ◽

10.1182/blood.v114.22.911.911 ◽

2009 ◽

Vol 114 (22) ◽

pp. 911-911 ◽

Cited By ~ 1

Author(s):

Martin Neumann ◽

Sandra Heesch ◽

Stefan Schwartz ◽

Nicola Gökbuget ◽

Dieter Hoelzer ◽

...

Keyword(s):

Gene Expression ◽

Stem Cell ◽

Real Time ◽

Gene Expression Profile ◽

Expression Profile ◽

Real Time Pcr ◽

Expression Profiles ◽

Lymphoblastic Leukemia ◽

Gene Expression Profiles ◽

Study Group

Abstract Abstract 911 Introduction: Recently, a small subgroup of pediatric acute T-lymphoblastic leukemia (T-ALL) was described, which is closely associated with the gene expression profile of early T-cell precursors (ETPs). This subtype, termed ETP-ALL, showed a highly unfavorable outcome compared to non-ETP(='typical')-ALL. Based on the results of Coustan-Smith et al. (Lancet Oncology, 2009), the Italian national study Associazione Italiana Ematologia Oncologia Pediatrica (AIEOP) and St-Jude Children's hospital modified their treatment in children with ETP-ALL to a more intensive regime including stem cell transplantation. ETP-ALL is characterized by a specific immunophenotype (CD1a-, CD8-, CD5weak with expression of stem cell or myeloid markers). Here we explored the existence of ETP-ALL in adults and further studied the molecular characteristics of this specific T-ALL subtype. Patients and methods: We examined the gene expression profiles of 86 adult T-ALL patients obtained from the Microarray Innovations in LEukemia (MILE) multicenter study (HG-U133 Plus 2.0, Affymetrix, Haferlach et al., JCO in press). In addition, bone marrow of 296 patients from the German Acute Lymphoblastic Leukemia Multicenter Study Group (GMALL) were analyzed by flow cytometry and expression levels of BAALC, IGFBP7, MN1, and WT1 were determined by real-time-PCR. Results: Using the published list of differentially expressed genes in ETPs (Coustan-Smith et al. 2009) we performed unsupervised clustering analyses of the 86 T-ALL samples. A cluster of 17 samples (19.8%) displayed an ETP-associated gene expression profile and were defined as ETP-ALL. Comparing the gene expression profiles of ETP-ALL and typical T-ALL, 2065 probe sets were differentially expressed in ETP-ALL (FDR 0.05). In addition to genes used for classification, we also identified genes known to be involved in the pathogenesis of T-ALL (e.g. PROM1, BCL2, LMO2, LYL1). In particular, stem cell associated genes such as, BAALC (2.52-fold, p=0.003), IGFBP7 (2.76-fold, p=0.002) or MN1 (3.41-fold, p<0.001) were upregulated in ETP-ALL, whereas HOX11 (45-fold, p=0.004), a marker for thymic T-ALL, was downregulated. An independent cohort of 297 patient samples from the GMALL study group was examined by flow cytometry and real-time PCR. 19 (6.4%) samples revealed the ETP-ALL immunophenotype. As expected, all patient samples were found in the group of early T-ALL, representing 23.5% of all early T-ALLs. There was a significant correlation between a lower leukocyte count at first diagnosis and the classification of ETP-ALL (p=0.001). Gene expression measured by real-time-PCR was performed for genes associated with poor outcome in T-ALL: BAALC (2.11-fold, p<0.001) and IGFBP7 (3.59-fold, p=0.003) were significantly upregulated in the group of ETP-ALL. Similarly, the genes MN1 (4.52-fold, p<0.001) and WT1 (2.76-fold, p=0.036), described as poor prognostic markers in cytogenetically normal AML, were also upregulated in ETP-ALL. Conclusion: In adult T-ALL, a subset of patients shares the gene expression profil and immunophenotype of ETP-ALL, which is in line with recent findings in pediatric patients. The gene expression profile of this subset is significantly correlated to stem cell associated markers predictive for inferior outcome in T-ALL. Interestingly, adverse factors in CN-AML are also aberrantly expressed in ETP-ALL suggesting a myeloid origin of ETPs and indicating a closer relationship between ETP-ALL and AML. The prognostic impact and the determination of the most appropiate set of markers needs to be further investigated. These results support the GMALL strategy to regard early T-ALL patients as high risk with assignment to stem cell transplantation. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Equity Ownership.

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Functional Somatic and Germline Variants in the NF-κB Pathway in Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v120.21.560.560 ◽

2012 ◽

Vol 120 (21) ◽

pp. 560-560 ◽

Cited By ~ 2

Author(s):

Ma. Reina Improgo ◽

Adam Kiezun ◽

Yaoyu Wang ◽

Lillian Werner ◽

Petar Stojanov ◽

...

Keyword(s):

Gene Expression ◽

Cell Death ◽

Chronic Lymphocytic Leukemia ◽

Transcriptional Activity ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Lymphocytic Leukemia ◽

Wild Type ◽

Rt Pcr ◽

Germline Variants

Abstract Abstract 560 Nuclear factor kappa B (NF-κB) encompasses a family of transcription factors involved in oncogenic processes including cellular proliferation and apoptotic inhibition. Constitutive activation of NF-κB has been observed in hematologic malignancies and is thought to confer resistance to chemotherapeutic agents. Here, we examine the role of the NF-κB pathway in chronic lymphocytic leukemia (CLL). Whole-exome sequencing was performed using tumor and matched germline DNA from 167 CLL patients. We identified 51 patients (30%) carrying 53 non-silent somatic variants in genes of the canonical NF-κB pathway, which consists of 272 genes as defined by the Ingenuity Pathway Analysis tool. Of the 99 patients whose germline sequences have been analyzed to date, 27 patients (27%) carry 34 non-silent germline variants in NF-κB pathway genes. A total of 67 patients (40%) have at least one non-silent somatic or germline variant. Variants in the NFKB1 gene, itself, were also observed: a somatic variant, H66R, found in two patients, and two germline variants, Y89F and R849W, each found in one patient. To evaluate the functional consequences of the NFKB1 variants, we performed site-directed mutagenesis to generate full-length NFKB1 cDNAs encoding these variants. We subsequently measured transcriptional activity of wild-type and mutant NFKB1 via luciferase assays in HEK293T cells using reporter cassettes containing the NFKB1 response element. Transcriptional activity of the three NFKB1 variants was found to be at least 2-fold higher than that of wild-type NFKB1 (p<0.0001). We further hypothesized that this increased transcriptional activity would lead to increased expression of NFKB1 downstream target genes. Analysis of gene expression profiles from Affymetrix HG-U133 Plus 2.0 Arrays of 65 CLL patient samples showed that the NFKB1 downstream targets CCL3, CCL4, and CD69 are upregulated in NFKB1 variants. To validate these results, we performed quantitative RT-PCR in patients with (n=3) or without (n=9) NFKB1 variants and confirmed upregulation of CCL3 (p=0.0286), CCL4 (p=0.0384), and CD69 (p=0.0263). Direct transfection of HEK293T cells with NFKB1 variants also resulted in a 3.3-fold upregulation of CCL3 (p=0.05). To test the hypothesis that deregulation of the NF-κB pathway is a key mechanism in CLL, we compared gene expression profiles of NF-κB pathway genes between CLL patient samples (n=146) and normal B cells (n=16) and found overall upregulation of the NF-κB pathway in CLL (Kolmogorov-Smirnov test, p=2.2e-16). K-means clustering and principal component analysis (PCA) further revealed that CLL patients can be divided into two subgroups exhibiting differential magnitude of NF-κB pathway upregulation. Studies in progress aim to identify the clinical significance of these subgroups. Finally, we assessed the effect of inhibiting the NF-κB pathway using the cell permeant NF-κB inhibitor, SN50. We performed Annexin V/PI staining 24 hours post-treatment in CLL cells with (n=9) or without (n=3) NF-κB pathway variants. SN50 increased cell death 1.8-fold in all cells tested (p<0.0001). Quantitative RT-PCR also showed a 59% decrease in expression of CCL3 one hour post-treatment, confirming inhibition of the NF-κB pathway. In conclusion, our findings demonstrate that a high proportion of CLL patients harbor somatic and germline variants in NF-κB pathway genes, some of which appear to be functional. Furthermore, the NF-κB pathway is upregulated in CLL and pharmacological inhibition of the pathway leads to increased cancer cell death. Functional characterization of NF-κB pathway variants offers mechanistic insight into the disease, providing novel targets for therapy. Disclosures: No relevant conflicts of interest to declare.

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Age-related changes in the transcriptional profile of mouse RPE/choroid

Physiological Genomics ◽

10.1152/physiolgenomics.00126.2003 ◽

2003 ◽

Vol 15 (3) ◽

pp. 258-262 ◽

Cited By ~ 28

Author(s):

Hisashi Ida ◽

Sharon A. Boylan ◽

Andrea L. Weigel ◽

Leonard M. Hjelmeland

Keyword(s):

Gene Expression ◽

Expression Profiles ◽

Cdna Probe ◽

Gene Expression Profiles ◽

Age Related Macular Degeneration ◽

Pcr Analysis ◽

Rt Pcr ◽

Mouse Cdna ◽

Age Related ◽

Age Related Changes

To evaluate the age-related changes in gene expression occurring in the complex of retinal pigmented epithelium, Bruch’s membrane, and choroid (RPE/choroid), we examined the gene expression profiles of young adult (2 mo) and old (24 mo) male C57BL/6 mice. cDNA probe sets from individual animals were synthesized using total RNA isolated from the RPE/choroid of each animal. Probes were amplified using the Clontech SMART system, radioactively labeled, and hybridized to two different Clontech Atlas mouse cDNA arrays. From each age group, three independent triplicates were hybridized to the arrays. Statistical analyses were performed using the Significance Analysis of Microarrays program (SAM version 1.13; Stanford University). Selected array results were confirmed by semi-quantitative RT-PCR analysis. Of 2,340 genes represented on the arrays, ∼60% were expressed in young and/or old mouse RPE/choroid. A moderate fraction (12%) of all expressed genes exhibited a statistically significant change in expression with age. Of these 150 genes, all but two, HMG14 and carboxypeptidase E, were upregulated with age. Many of these upregulated genes can be grouped into several broad functional categories: immune response, proteases and protease inhibitors, stress response, and neovascularization. RT-PCR results from six of six genes examined confirmed the differential change in expression with age of these genes. Our study provides likely candidate genes to further study their role in the development of age-related macular degeneration and other aging diseases affecting the RPE/choroid.

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