High-Throughput Real-Time PCR for Detection of Gene-Expression Levels

2009 ◽  
pp. 167-175 ◽  
Author(s):  
Bridget K. Wagner ◽  
Zoltan Arany
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
High Throughput ◽  
Real Time Pcr ◽  
Expression Levels ◽  
Gene Expression Levels

Discrimination and evaluation of lactoferrin and delta-lactoferrin gene expression levels in cancer cells and under inflammatory stimuli using TaqMan real-time PCR

BioMetals ◽  
2010 ◽  
Vol 23 (3) ◽  
pp. 441-452 ◽  
Author(s):  
Esthelle Hoedt ◽  
Stephan Hardivillé ◽  
Christophe Mariller ◽  
E. Elass ◽  
Jean-Paul Perraudin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cancer Cells ◽  
Real Time ◽  
Real Time Pcr ◽  
Expression Levels ◽  
Inflammatory Stimuli ◽  
Lactoferrin Gene ◽  
Gene Expression Levels

scholarly journals Lack of correlation between in silico projection of function and quantitative real-time PCR-determined gene expression levels in colon tissue

2013 ◽  
pp. 99
Author(s):  
Susan Kadlubar ◽  
Rosalind Penney ◽  
Abbie Lundgreen ◽  
Aiwei Yao-Borengassar ◽  
Vineetha Koroth-Edavana ◽  
...  
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Real Time Pcr ◽  
In Silico ◽  
Quantitative Real Time Pcr ◽  
Colon Tissue ◽  
Expression Levels ◽  
Gene Expression Levels

scholarly journals High-throughput mRNA sequencing of stromal cells from endometriomas and endometrium

Reproduction ◽  
2017 ◽  
Vol 154 (1) ◽  
pp. 93-100 ◽  
Author(s):  
Kadri Rekker ◽  
Merli Saare ◽  
Elo Eriste ◽  
Tõnis Tasa ◽  
Viktorija Kukuškina ◽  
...  
Keyword(s):  
Gene Expression ◽  
High Throughput ◽  
Complement System ◽  
Stromal Cells ◽  
Cell Types ◽  
Coagulation Cascade ◽  
Expression Levels ◽  
Mrna Sequencing ◽  
Standard Design ◽  
Gene Expression Levels

The aetiology of endometriosis is still unclear and to find mechanisms behind the disease development, it is important to study each cell type from endometrium and ectopic lesions independently. The objective of this study was to uncover complete mRNA profiles in uncultured stromal cells from paired samples of endometriomas and eutopic endometrium. High-throughput mRNA sequencing revealed over 1300 dysregulated genes in stromal cells from ectopic lesions, including several novel genes in the context of endometriosis. Functional annotation analysis of differentially expressed genes highlighted pathways related to cell adhesion, extracellular matrix–receptor interaction and complement and coagulation cascade. Most importantly, we found a simultaneous upregulation of complement system components and inhibitors, indicating major imbalances in complement regulation in ectopic stromal cells. We also performed in vitro experiments to evaluate the effect of endometriosis patients’ peritoneal fluid (PF) on complement system gene expression levels, but no significant impact of PF on C3, CD55 and CFH levels was observed. In conclusion, the use of isolated stromal cells enables to determine gene expression levels without the background interference of other cell types. In the future, a new standard design studying all cell types from endometriotic lesions separately should be applied to reveal novel mechanisms behind endometriosis pathogenesis.

Clinical Measurement of Prognostic Immune Signature in Follicular Lymphoma by RT-PCR Based Gene Expression Profiling and Immunohistochemistry Demonstrates Favourable T-Cell and Unfavorable Macrophage Infiltration.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 359-359
Author(s):  
Richard J. Byers ◽  
Ebrahim Sakhinia ◽  
Preethi Joseph ◽  
Caroline Glennie ◽  
Sara McDermott ◽  
...  
Keyword(s):  
Gene Expression ◽  
Immune Response ◽  
Image Analysis ◽  
Real Time ◽  
High Power ◽  
Real Time Pcr ◽  
High Power Field ◽  
Gene Signatures ◽  
Expression Levels ◽  
Survival Interval

Abstract Gene expression profiling studies have demonstrated immune response gene signatures predictive of outcome in follicular lymphoma (FL) and there is a need for validation of these signatures and for their translation to clinical use. However, measurement of these genes in routine practice remains difficult and to date there have been very few studies validating the hypothesis. We have previously demonstrated the utility of real-time PCR measurement of gene expression levels in globally amplified polyA cDNA as a clinically practical method for translation of gene signatures to clinical use. In this project we extended the method to analysis of immune response signatures in FL. We used real-time PCR to measure expression levels (normalised to the mean of 4 housekeeping genes) of 36 candidate Indicator genes, selected from microarray studies, in polyA cDNAs prepared using polyA PCR (method detailed in Sakhinia et al 2007) from 58 archived human frozen lymph nodes, together with immunohistochemistry for CD3, CD4, CD7, CD8, CD10, CD20, CD21 and CD68 in parallel formalin fixed paraffin embedded tissue samples to measure immune response in FL. Immunohistochemical positivity was measured by a semi-automated image analysis method using spectral unmixing to identify areas of immunopositivity. Kaplan-Mier survival analysis was performed against the normalised real-time PCR expression levels of each of the genes and against the percentage immunohistochemical postivity for CD3, CD4, CD7, CD8, CD10, CD20, CD21; for CD68 survival analysis was performed for cases with either 15 or less or more than 15 CD68 positive cells per high power field (hpf). High levels of CCR1, a marker of monocyte actication, were associated with a shorter survival interval (p<0.02) (figure 1a), whilst immunohistochemistry demonstrated association of high numbers of CD7 positive T-cells with longer survival interval (figure 1b) (p<0.032) and of high numbers of CD68 positive macrophages with a shorter survival interval (figure 1c) (p<0.02). The results confirm the role of the host immune response in outcome in FL and identify CCR1 as a prognostic indicator and marker of immune switch between macrophage and T-cell dominant response. The methods used are clinically applicable, whilst the clinical utility of polyA DNA and real-time PCR for measurement of gene signatures and the strength of this approach as a “molecular block” are confirmed. Kaplan-Meier Survival Plots for upper (3&4) and lower (1&2) quartiles of a) CCR1 expression and b) number of CD7 +ve cells, and c) cases with less then vs greater than or equal to 15 macrophages per high power field CCR1 measured by real-time PCR and CD7 and macrophage numbers by immunohistochemistry and image analysis Kaplan-Meier Survival Plots for upper (3&4) and lower (1&2) quartiles of a) CCR1 expression and b) number of CD7 +ve cells, and c) cases with less then vs greater than or equal to 15 macrophages per high power field CCR1 measured by real-time PCR and CD7 and macrophage numbers by immunohistochemistry and image analysis

scholarly journals Gene expression patterns in bone following lipopolysaccharide stimulation

2014 ◽  
Vol 19 (4) ◽  
Author(s):  
Jing Yang ◽  
Nan Su ◽  
Xiaolan Du ◽  
Lin Chen
Keyword(s):  
Gene Expression ◽  
Bone Formation ◽  
Real Time ◽  
Real Time Pcr ◽  
Early Stage ◽  
System Development ◽  
Expression Patterns ◽  
Osteoclast Differentiation ◽  
Gene Expression Patterns ◽  
Expression Levels

AbstractBone displays suppressed osteogenesis in inflammatory diseases such as sepsis and rheumatoid arthritis. However, the underlying mechanisms have not yet been clearly explained. To identify the gene expression patterns in the bone, we performed Affymetrix Mouse Genome 430 2.0 Array with RNA isolated from mouse femurs 4 h after lipopolysaccharide (LPS) administration. The gene expressions were confirmed with real-time PCR. The serum concentration of the N-terminal propeptide of type I collagen (PINP), a bone-formation marker, was determined using ELISA. A total of 1003 transcripts were upregulated and 159 transcripts were downregulated (more than twofold upregulation or downregulation). Increased expression levels of the inflammation-related genes interleukin-6 (IL-6), interleukin-1β (IL-1β) and tumor necrosis factor α (TNF-α) were confirmed from in the period 4 h to 72 h after LPS administration using real-time PCR. Gene ontogene analysis found four bone-related categories involved in four biological processes: system development, osteoclast differentiation, ossification and bone development. These processes involved 25 upregulated genes. In the KEGG database, we further analyzed the transforming growth factor β (TGF-β) pathway, which is strongly related to osteogenesis. The upregulated bone morphogenetic protein 2 (BMP2) and downregulated inhibitor of DNA binding 4 (Id4) expressions were further confirmed by real-time PCR after LPS stimulation. The osteoblast function was determined through examination of the expression levels of core binding factor 1 (Cbfa1) and osteocalcin (OC) in bone tissues and serum PINP from 4 h to 72 h after LPS administration. The expressions of OC and Cbfa1 decreased 6 h after administration (p < 0.05). Significantly suppressed PINP levels were observed in the later stage (from 8 h to 72 h, p < 0.05) but not in the early stage (4 h or 6 h, p > 0.05) of LPS stimulation. The results of this study suggest that LPS induces elevated expressions of skeletal system development- and osteoclast differentiation-related genes and inflammation genes at an early stage in the bone. The perturbed functions of these two groups of genes may lead to a faint change in osteogenesis at an early stage of LPS stimulation. Suppressed bone formation was found at later stages in response to LPS stimulation.

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