RT-PCR-Based Gene Expression Profiling for Cancer Biomarker Discovery from Fixed, Paraffin-Embedded Tissues

2011 ◽  
pp. 239-257 ◽  
Author(s):  
Aaron Scott ◽  
Ranjana Ambannavar ◽  
Jennie Jeong ◽  
Mei-Lan Liu ◽  
Maureen T. Cronin
Keyword(s):  
Gene Expression ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Biomarker Discovery ◽  
Cancer Biomarker ◽  
Rt Pcr

scholarly journals 256CONSTRUCTION OF A BOVINE CDNA ARRAY TO MONITOR GENE EXPRESSION PROFILES IN BOVINE PREIMPLANTATION EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 248
Author(s):  
C. Wrenzycki ◽  
T. Brambrink ◽  
D. Herrmann ◽  
J.W. Carnwath ◽  
H. Niemann
Keyword(s):  
Gene Expression ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Expression Profiles ◽  
Cdna Array ◽  
Preimplantation Embryos ◽  
Average Insert Size ◽  
Rt Pcr ◽  
Public Data

Array technology is a widely used tool for gene expression profiling, providing the possibility to monitor expression levels of an unlimited number of genes in various biological systems including preimplantation embryos. The objective of the present study was to develop and validate a bovine cDNA array and to compare expression profiles of embryos derived from different origins. A bovine blastocyst cDNA library was generated. Poly(A+)RNA was extracted from in vitro-produced embryos using a Dynabead mRNA purification kit. First-strand synthesis was performed with SacIT21 primer followed by randomly primed second-strand synthesis with a DOP primer mix (Roche) and a global PCR with 35 cycles using SacIT21 and DOP primers. Complementary DNA fragments from 300 to 1500bp were extracted from the gel and normalized via reassoziation and hydroxyapatite chromatography. Resulting cDNAs were digested with SacI and XhoI, ligated into a pBKs vector, and transfected into competent bacteria (Stratagene). After blue/white colony selection, plasmids were extracted and the inserts were subjected to PCR using vector specific primers. Average insert size was determined by size idenfication on agarose gels stained with ethidium bromide. After purification via precipitation and denaturation, 192 cDNA probes were double-spotted onto a nylon membrane and bound to the membrane by UV cross linking. Amplified RNA (aRNA) probes from pools of three or single blastocysts were generated as described recently (Brambrink et al., 2002 BioTechniques, 33, 3–9) and hybridized to the membranes. Expression profiles of in vitro-produced blastocysts cultured in either SOF plus BSA or TCM plus serum were compared with those of diploid parthenogenetic ones generated by chemical activation. Thirty-three probes have been sequenced and, after comparison with public data bases, 26 were identified as cDNAs or genes. Twelve out of 192 (6%) seem to be differentially expressed within the three groups;; 7/12 (58%) were down-regulated, 3/12 (25%) were up-regulated in SOF-derived embryos, and 2/12 (20%) were up-regulated in parthenogenetic blastocysts compared to their in vitro-generated counterparts. Three of these genes involved in calcium signaling (calmodulin, calreticulin) and regulation of actin cytoskeleton (destrin) were validated by semi-quantitative RT-PCR (Wrenzycki et al., 2001 Biol. Reprod. 65, 309–317) employing poly(A+) RNA from a single blastocyst as starting material. No differences were detected in the relative abundance of the analysed gene transcripts within the different groups. These findings were confirmed employing the aRNA used for hybridization in RT-PCR and showed a good representativity of the selected transcripts. Results indicate that it is possible to construct a homologous cDNA array which could be used for gene expression profiling of bovine preimplantation embryos. Supported by the Deutsche Forschungsgemeinschaft (DFG Ni 256/18-1).

Global gene expression profiling in early-stage polycystic kidney disease in the Han:SPRD Cy rat identifies a role for RXR signaling

2011 ◽  
Vol 300 (1) ◽  
pp. F177-F188 ◽  
Author(s):  
Masanori Kugita ◽  
Kazuhiro Nishii ◽  
Miwa Morita ◽  
Daisuke Yoshihara ◽  
Hiroe Kowa-Sugiyama ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Proliferation ◽  
Kidney Disease ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Early Stage ◽  
Global Gene Expression ◽  
Rt Pcr ◽  
Polycystic Kidney ◽  
Global Gene Expression Profiling

Han:SPRD Cy is a spontaneous rat model of polycystic kidney disease (PKD) caused by a missense mutation in Pkdr1. Cystogenesis in this model is not clearly understood. In the current study, we performed global gene expression profiling in early-stage PKD cyst development in Cy/Cy kidneys and normal (+/+) kidneys at 3 and 7 days of postnatal age. Expression profiles were determined by microarray analysis, followed by validation with real-time RT-PCR. Genes were selected with over 1.5-fold expression changes compared with age-matched +/+ kidneys for canonical pathway analysis. We found nine pathways in common between 3- and 7-day Cy/Cy kidneys. Three significantly changed pathways were designated “Vitamin D Receptor (VDR)/Retinoid X Receptor (RXR) Activation,” “LPS/IL-1-Mediated Inhibition of RXR Function,” and “Liver X Receptor (LXR)/RXR Activation.” These results suggest that RXR-mediated signaling is significantly altered in developing kidneys with mutated Pkdr1. In gene ontology analysis, the functions of these RXR-related genes were found to be involved in regulating cell proliferation and organ morphogenesis. With real-time RT-PCR analysis, the upregulation of Ptx2, Alox15b, OSP, and PCNA, major markers of cell proliferation associated with the RXR pathway, were confirmed in 3- and 7-day Cy/Cy kidneys compared with 3-day +/+ kidneys. The increased RXR protein was observed in both the nucleus and cytoplasm of cystic epithelial cells in early-stage Cy/Cy kidneys, and the RXR-positive cells were strongly positive for PCNA staining. Taken together, cell proliferation and organ morphogenesis signals transduced by RXR-mediated pathways may have important roles for cystogenesis in early-stage PKD in this Pkdr1-mutated Cy rat.

Gene Expression Profiling of Peripheral T-Cell Lymphoma (PTCL, NOS) and Comparison to Diffuse Large B-Cell Lymphoma (DLBCL).

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2277-2277
Author(s):  
Daruka Mahadevan ◽  
Catherine Spier ◽  
Kimiko Della Croce ◽  
Susan Miller ◽  
Benjamin George ◽  
...  
Keyword(s):  
Gene Expression ◽  
Lymph Node ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Gene Expression Profiling ◽  
Real Time ◽  
Expression Profiling ◽  
Cell Lymphoma ◽  
Rt Pcr

Abstract Background: WHO classifies NHL into B (~85%) and T (~15%) cell subtypes. Of the T-cell NHL, peripheral T-cell NHL (PTCL, NOS) comprises ~6–10% with an inferior response and survival to chemotherapy compared to DLBCL. Gene Expression Profiling (GEP) of DLBCL has provided molecular signatures that define 3 subclasses with distinct survival rates. The current study analyzed transcript profiling in PTCL (NOS) and compared and contrasted it to GEP of DLBCL. Methods : Snap frozen samples of 5 patients with PTCL (NOS) and 4 patients with DLBCL were analyzed utilizing the HG-U133A 2.0 Affymetrix array (~18,400 transcripts, 22,000 probe sets) after isolating and purifying total RNA (Qiagen, RNAeasy). The control RNA samples were isolated from normal peripheral blood (PB) B-cell (AllCell, CA), normal PB T-cell (AllCell, CA) and normal lymph node (LN). Immunohisto-chemistry (IHC) confirmed tumor lineage and quantitative real time RT-PCR was performed on selected genes to validate the microarray study. The GEP data were processed and analyzed utilizing Affymetrix MAS 5.0 and GeneSpring 5.0 software. Our data were analyzed in the light of the published GEP of DLBCL (lymphochip and affymtrix) and the validated 10 prognostic genes (by IHC and real time RT-PCR). Results : Data are represented as “robust” increases or decreases of relative gene expression common to all 5 PTCL or 4 DLBCL patients respectively. The table shows the 5 most over-expressed genes in PTCL or DLBCL compared to normal T-cell (NT), B-cell (NB) and lymph node (LN). PTCL vs NT PTCL vs LN DLVCL vs NB DLBCL vs LN COL1A1 CHI3L1 CCL18 CCL18 CCL18 CCL18 VNN1 IGJ CXCL13 CCL5 UBD VNN1 IGFBP7 SH2D1A LYZ CD52 RARRES1 NKG7 CCL5 MAP4K1 Of the top 20 increases, 3 genes were common to PTCL and DLBCL when compared to normal T and B cells, while 11 were common when compared to normal LN. Comparison of genes common to normal B-cell and LN Vs DLBCL or PTCL and normal T-cell and LN Vs PTCL or DLBCL identified sets of genes that are commonly and differentially expressed in PTCL and/or DLBCL. The 4 DLBCL patients analyzed express 3 of 10 prognostic genes compared to normal B-cells and 7 of 10 prognostic genes compared to normal LN and fall into the non-germinal center subtype. Quantitative real time RT-PCR on 10 functionally distinct common over-expressed genes in the 5 PTCL (NOS) patients (Lumican, CCL18, CD14, CD54, CD106, CD163, α-PDGFR, HCK, ABCA1 and Tumor endothelial marker 6) validated the microarray data. Conclusions: GEP of PTCL (NOS) and DLBCL in combination with quantitative real time RT-PCR and IHC have identified a ‘molecular signature’ for PTCL and DLBCL based on a comparison to normal (B-cell, T-cell and LN) tissue. The categorization of the GEP based on the six hallmarks of cancer identifies a ‘tumor profile signature’ for PTCL and DLBCL and a number of novel targets for therapeutic intervention.

scholarly journals Utility of the Blood for Gene Expression Profiling and Biomarker Discovery in Chronic Fatigue Syndrome

2002 ◽  
Vol 18 (4) ◽  
pp. 193-199 ◽  
Author(s):  
Suzanne D. Vernon ◽  
Elizabeth R. Unger ◽  
Irina M. Dimulescu ◽  
Mangalathu Rajeevan ◽  
William C. Reeves
Keyword(s):  
Gene Expression ◽  
Chronic Fatigue Syndrome ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Biomarker Discovery ◽  
Chronic Fatigue ◽  
Classification Algorithms ◽  
Control Analysis ◽  
Healthy Controls ◽  
Fatigue Syndrome

Chronic fatigue syndrome (CFS) is a debilitating illness lacking consistent anatomic lesions and eluding conventional laboratory diagnosis. Demonstration of the utility of the blood for gene expression profiling and biomarker discovery would have implications into the pathophysiology of CFS. The objective of this study was to determine if gene expression profiles of peripheral blood mononuclear cells (PMBCs) could distinguish between subjects with CFS and healthy controls. Total RNA from PBMCs of five CFS cases and seventeen controls was labeled and hybridized to 1764 genes on filter arrays. Gene intensity values were analyzed by various classification algorithms and nonparametric statistical methods. The classification algorithms grouped the majority of the CFS cases together, and distinguished them from the healthy controls. Eight genes were differentially expressed in both an age-matched case-control analysis and when comparing all CFS cases to all controls. Several of the diffrentially expressed genes are associated with immunologic functions (e.g., CMRF35 antigen, IL-8, HD protein) and implicate immune dysfunction in the pathophysiology of CFS. These results successfully demonstrate the utility of the blood for gene expression profiling to distinguish subjects with CFS from healthy controls and for identifying genes that could serve as CFS biomarkers.

Identification of New Megakaryocytic Subpopulations in Mouse Bone Marrow.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2265-2265
Author(s):  
Shinji Sogo ◽  
Kuniko Matsumura-Takeda ◽  
Yoshimasa Isakari ◽  
Yasuo Harada ◽  
Kinue Nishioka ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Molecular Mechanisms ◽  
Present Finding ◽  
The Other ◽  
Mouse Bone Marrow ◽  
Rt Pcr ◽  
Kit Gene

Abstract Platelets (PLT) are produced from megakaryocytes (Mks) via proplatelet formation (PPF). However, the molecular mechanisms from Mks to PPF are not clearly elucidated, because the maturational steps of the Mks in bone marrow (BM) are not analyzed in detail. Until now, mouse Mks have been only isolated as acetylcholinesterase (AchE) positive cells and they are understood as well maturated population. In this study, we found the presence of different megakaryocytic subpopulations in BM by flowcytometry. To isolate the Mks, first we depleted lineage marker (CD4, CD8a, CD11b, B220, CD71, CD90, TER119, Gr-1, F4/80, 7/4) positive cells from BM cells of BALB/c mice. The analysis of the expression-pattern of CD41, CD45 and CD61 in the lineage negative (Lin−) cells showed the presence of two types of megakaryocytic subpopulations. By sorting, they were identified as Lin−CD41+/45+/61+ cells (AchE negative) and Lin−CD41++/45+/61++ cells (partially AchE positive), respectively. To assess the maturational stages of the subpopulations, each population was cultured with 10ng/mL of TPO followed by counting of PPF and PLT production. Both PPF and PLT production were observed in Lin−CD41+/45+/61+ cells later than those in Lin−CD41++/45+/61++ cells. On the other hand, CFU-Mk was scarcely detected in each subpopulation. The results indicate that both populations are the committed megakaryocytes and Lin−CD41+/45+/61+ cells are more immature population than Lin−CD41++/45+/61++ cells. Then to characterize these subpopulations in detail, gene expression profiling was performed against four-megakaryocytic lineage-populations, Lin−CD41−Thy1lowc-kit+ cells as stem/progenitor, Lin−CD41+/45+/61+ cells, Lin−CD41++/45+/61++ cells and PLT using GeneChipU74 or RT-PCR. These analyses revealed that many PLT-specific genes including gpIb/IX, P-selectin, thrombin-R and ADP-R were already expressed on Lin−CD41+/45+/61+ cells but less than Lin−CD41++/45+/61++ cells. Especially, beta-1 tubulin that is necessary for PPF was only expressed on Lin−CD41++/45+/61++ cells. On the contrary, the expression of c-kit gene was gradually decreasing from stem/progenitor fraction to PLT. In conclusion, we succeeded in the isolation of new subpopulations distinguishable between immature Mks and more matured Mks beginning to prepare PLT. The present finding can contribute to elucidate the molecular mechanisms during terminal maturation.

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