Customer Feature


Dr. Triche Speaks About His Research Focus and NuGEN Solution

Dr. Timothy J. Triche is a professor in the Pathology, Cancer Biology, and Pediatrics Departments at The Saban Research Institute of the Childrens Hospital Los Angeles





"My research focuses on genetic aspects of cancer and the implications for improved diagnosis and therapy.  Current research emphasizes whole genome approaches to gene expression profiling and genomic structural alterations using very high density microarrays.  A major impediment to broader application of our studies is the lack of fresh frozen tumor tissue for our clinical correlative studies.  In the real world of tumor diagnosis and estimation of both prognosis and therapeutic responsiveness, the only widely available specimen is formalin fixed, paraffin embedded tissue (FFPE).  The challenge has been to adapt these specimens to the rigorous and complex analyses we currently undertake on them.

Our ultimate goal is to learn from ideal, fresh frozen tumor tissue and apply the important 'biomarker' information gleaned there from to the less than ideal but still highly usable FFPE specimens that are universally available everywhere.  We are encouraged with our progress to date using the NuGEN amplification technology on these suboptimal specimens for true global RNA expression profiling, our current area of particular interest.

We have recently undertaken a broad validation study to assess the overall performance, accuracy and reproducibility of expression measures obtained with the Affymetrix GeneChip® U133 Plus 2.0 arrays as well as the GeneChip Human Exon 1.0 ST array platform.  Our goal is to demonstrate the utility for the Exon platform in biomarker validation as well as discovery.

Using the NuGEN WT-Ovation™ FFPE System for target preparation, we have completed the first stage of our study on the U133 Plus 2.0 arrays using a matched set of paired fresh-frozen and FFPE-archived tissue specimens of various age from a Wilm's tumor and the normal adjacent kidney tissue.  The NuGEN assays yielded samples with reliable expression levels in FFPE compared with snap frozen samples, delivering the desired accuracy and sensitivity.  Through an active collaboration with the NuGEN research team, using the NuGEN FFPE System as well as the WT-Ovation™ Exon Module, we are in the process of completing the second stage, assessing and optimizing assay performance on Human Exon arrays. 

To our knowledge, this study is the first attempt to assess quantitative gene expression analysis of FFPE material using Affymetrix Human Exon 1.0 ST arrays.  A manuscript is in preparation summarizing the results of this study and providing guidelines for molecular profiling of clinical samples from FFPE material."


Researchers Meet Gene Expression Analysis Challenges With NuGEN Solutions

Gene expression analysis is a valuable tool in understanding complex biological mechanisms. To obtain high resolution and unambiguous expression profiles, many studies require isolation of homogeneous cell populations using technologies such as laser captured microdissections (LCM) and Fluorescent Activated Cell Sorting (FACS).    The challenge with using these highly selected cell populations derived from very limited biological specimens is obtaining sufficient amounts of sample for in-depth validation of known signatures and expression profiles.

Publications such as those featured below by Guo et al, and Pahler et al demonstrate how researchers who use qPCR analysis overcome these challenges by employing the NuGEN pre-amplification approach.  In both studies, investigators isolated small, homogeneous populations of cells by Fluorescent Activated Cell Sorting (FACS) and isolated total RNA from these limited cell suspensions. The RNA was subsequently amplified with the NuGEN WT-Ovation RNA Amplification System to generate microgram quantities of cDNA prior to performing gene expression analysis using qPCR.
 
Benefits of the NuGEN whole transcriptome pre-amplification approach include:

-  Enabling analysis of small and limited samples across a wide range of transcript abundance, resulting in higher sensitivity and reproducibility.

-  Allowing high throughput qPCR analysis by providing sufficient sample amounts.

-  Enabling analysis of compromised samples such as biopsies, fine needle aspirates, micro-dissections, etc., despite the typical loss of transcript 3' ends due to degradation.

-  Providing flexibility in qPCR probe design across the entire transcript.

-  Allowing robust qPCR experimental designs that require triplicate reactions and several types of controls, and therefore larger amounts of sample.

-  Providing cDNA, a more stable and consistent sample for future analysis without depleting the original RNA samples.

-  Facilitating sharing samples with collaborators and consortia members.

Want to learn more?  Read the WT-Ovation™ Amplification System Bulletin #2: Frequently Asked Questions on RNA Amplification Prior to qPCR


Publications of the Month

Plasticity in Tumor-Promoting Inflammation: Impairment of Macrophage Recruitment Evokes a Compensatory Neutrophil Response
Jessica C Pahler, Simon Tazzyman, Neta Erez, Yung-Yi Chen, Craig Murdoch, Hiroaki Nozawa, Claire E Lewis, and Douglas Hanahan

Neoplasia. 2008 April; 10(4): 329-339; PMCID: PMC2288539

Abstract
Previous studies in the K14-HPV/E2 mouse model of cervical carcinogenesis demonstrated that infiltrating macrophages are the major source of matrix metalloproteinase 9 (MMP-9), a metalloprotease important for tumor angiogenesis and progression.  We observed increased expression of the macrophage chemoattractant, CCL2, and its receptor, CCR2, concomitant with macrophage influx and MMP-9 expression.  To study the role of CCL2-CCR2 signaling in cervical tumorigenesis, we generated CCR2-deficient K14-HPV/E2 mice.  Cervixes of CCR2-null mice contained significantly fewer macrophages.

Surprisingly, there was only a modest delay in time to progression from dysplasia to carcinoma in the CCR2-deficient mice, and no difference in end-stage tumor incidence or burden.  Moreover, there was an unexpected persistence of MMP-9 activity, associated with increased abundance of MMP-9+ neutrophils in tumors from CCR2-null mice.  In vitro bioassays revealed that macrophages produce soluble factor(s) that can suppress neutrophil dynamics, as evidenced by reduced chemotaxis in response to CXCL8, and impaired invasion into three-dimensional tumor masses grown in vitro.  Our data suggest a mechanism whereby CCL2 attracts proangiogenic CCR2+ macrophages with the ancillary capability to limit infiltration by neutrophils.  If such tumor-promoting macrophages are suppressed, MMP-9+ neutrophils are then recruited, providing alternative paracrine support for tumor angiogenesis and progression.


CD4+CD25+ Regulatory T Cells in the Small Intestinal Lamina Propria Show an Effector/Memory Phenotype
Zijin Guo, Myoung Ho Jang, Kazuhiro Otani, Zhongbin Bai, Eiji Umemoto, Masanori Matsumoto, Mika Nishiyama, Mikako Yamasaki, Satoshi Ueha, Kouji Matsushima, Takako Hirata and Masayuki Miyasaka

International Immunology, published online on January 9, 2008 doi:10.1093/intimm/dxm143.
(c) The Author 2008. Published by Oxford University Press on behalf of The Japanese Society for Immunology. All rights reserved.

Abstract
CD4+CD25+ regulatory T cells (Tregs) have been implicated in the suppression of pathogenic responses to both self- and non-self-antigens in the intestine. However, their precise properties and functions in the gut, as well as the molecular basis of their recruitment to the gut, are poorly understood.  Here, we found that most of the CD4+CD25+ T cells in the small intestinal lamina propria (LP) express Foxp3 and exhibit an 'effector/memory' phenotype, CD44hiCD45RBloCD62L-, whereas only a minority of the Foxp3+CD4+CD25+ T cells in the spleen and mesenteric lymph nodes showed this phenotype.  The Tregs in the small intestinal LP (LP-Tregs) expressed higher levels of CCR4 and CCR9 and a substantially lower level of CCR7 than the Tregs in the spleen. In vitro, the LP-Tregs showed chemotaxis to CCL25/thymus-expressed chemokine.  In addition, they showed efficient chemotaxis to the CCR4 ligands, CCL17/thymus and activation-regulated chemokine and CCL22/macrophage-derived chemokine, which are abundantly expressed by dendritic cells (DCs) in the small intestinal LP.  In vivo,  50% of the LP-Tregs were closely associated or in direct contact with LP-DCs.  These findings demonstrate that LP-Tregs are phenotypically and functionally unique and raise the possibility that they are retained in the small intestinal LP through the action of CCL17 and CCL22, which are locally produced by LP-DCs.


Attending the Atlantic Omics Symposium and Expo in Canada?Stop by the BioLynx booth and learn about NuGEN Solutions
Delta Beausejour, Moncton, New Brunswick, Canada, August 19-20, 2008

Organized by the Atlantic Cancer Research Institute (ACRI) and the Atlantic Microarray Facility (AMF), in collaboration with the National Research Council Canada, this two-day symposium in, will serve as a forum for exploring specific interests in the Omics.  The event will feature four keynote speakers, a host of international presenters, posters and vendor exhibitions.


Ovation Systems Family of RNA Amplification and Labeling Products

The NuGEN Ovation® Systems family of RNA amplification and labeling products enables sensitive, robust gene expression profiling and novel signature discovery using a variety of challenging biological samples such as FFPE, whole blood, LCM, fine needle aspirates, tissue biopsies, sorted cell, and more.  The flexible modular products let you chose your preferred analytical platform;  Affymetrix® 3-prime or Exon, Agilent, Illumina® microarrays, or qPCR. 


If this message has been forwarded to you, you may join our mailing list.

NuGEN Technologies, Inc.
821 Industrial Road, Unit A
San Carlos, CA 94070
Telephone: 888-654-6544 or 650-590-3600
www.nugeninc.com