Rapid quantification of murine endothelin-1 and vasoactive intestinal contractor gene expression levels by a real-time PCR system

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

Download Full-text

Gene expression patterns in bone following lipopolysaccharide stimulation

Cellular & Molecular Biology Letters ◽

10.2478/s11658-014-0216-2 ◽

2014 ◽

Vol 19 (4) ◽

Cited By ~ 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.

Download Full-text

Telomerase Activity and Telomerase Subunit Gene Expression Levels Are Not Related in Prostate Cancer: A Real-Time Quantification and In Situ Hybridization Study

Laboratory Investigation ◽

10.1097/01.lab.0000069035.85309.30 ◽

2003 ◽

Vol 83 (5) ◽

pp. 623-633 ◽

Cited By ~ 17

Author(s):

Joern Kamradt ◽

Carsten Drosse ◽

Sascha Kalkbrenner ◽

Volker Rohde ◽

Ramona Lensch ◽

...

Keyword(s):

Gene Expression ◽

Prostate Cancer ◽

In Situ Hybridization ◽

Real Time ◽

Telomerase Activity ◽

Hybridization Study ◽

Subunit Gene ◽

Expression Levels ◽

Gene Expression Levels

Download Full-text

Laser Capture Microdissection and Real-Time PCR for Analysis of Glomerular Endothelin-1 Gene Expression in Mesangiolysis of Rat Anti-Thy 1.1 and Murine Habu Snake Venom Glomerulonephritis

Diagnostic Molecular Pathology ◽

10.1097/00019606-200306000-00007 ◽

2003 ◽

Vol 12 (2) ◽

pp. 108-117 ◽

Cited By ~ 14

Author(s):

A. Dimmler ◽

C. S. Haas ◽

S. Cho ◽

M. Hattler ◽

C. Forster ◽

...

Keyword(s):

Gene Expression ◽

Real Time ◽

Snake Venom ◽

Laser Capture Microdissection ◽

Real Time Pcr ◽

Endothelin 1 ◽

Laser Capture

Download Full-text

Selection of reference genes for quantitative real-time PCR analysis in cucumber (Cucumis sativus L.), pumpkin (Cucurbita moschata Duch.) and cucumber–pumpkin grafted plants

PeerJ ◽

10.7717/peerj.6536 ◽

2019 ◽

Vol 7 ◽

pp. e6536 ◽

Cited By ~ 5

Author(s):

Li Miao ◽

Xing Qin ◽

Lihong Gao ◽

Qing Li ◽

Shuzhen Li ◽

...

Keyword(s):

Gene Expression ◽

Real Time ◽

Reference Gene ◽

Real Time Pcr ◽

Reference Genes ◽

Stable Expression ◽

Quantitative Real Time Pcr ◽

Graft Union ◽

Expression Levels ◽

Qrt Pcr

Background Quantitative real-time PCR (qRT-PCR) is a commonly used high-throughput technique to measure mRNA transcript levels. The accuracy of this evaluation of gene expression depends on the use of optimal reference genes. Cucumber–pumpkin grafted plants, made by grafting a cucumber scion onto pumpkin rootstock, are superior to either parent plant, as grafting conveys many advantages. However, although many reliable reference genes have been identified in both cucumber and pumpkin, none have been obtained for cucumber–pumpkin grafted plants. Methods In this work, 12 candidate reference genes, including eight traditional genes and four novel genes identified from our transcriptome data, were selected to assess their expression stability. Their expression levels in 25 samples, including three cucumber and three pumpkin samples from different organs, and 19 cucumber–pumpkin grafted samples from different organs, conditions, and varieties, were analyzed by qRT-PCR, and the stability of their expression was assessed by the comparative ΔCt method, geNorm, NormFinder, BestKeeper, and RefFinder. Results The results showed that the most suitable reference gene varied dependent on the organs, conditions, and varieties. CACS and 40SRPS8 were the most stable reference genes for all samples in our research. TIP41 and CACS showed the most stable expression in different cucumber organs, TIP41 and PP2A were the optimal reference genes in pumpkin organs, and CACS and 40SRPS8 were the most stable genes in all grafted cucumber samples. However, the optimal reference gene varied under different conditions. CACS and 40SRPS8 were the best combination of genes in different organs of cucumber–pumpkin grafted plants, TUA and RPL36Aa were the most stable in the graft union under cold stress, LEA26 and ARF showed the most stable expression in the graft union during the healing process, and TIP41 and PP2A were the most stable across different varieties of cucumber–pumpkin grafted plants. The use of LEA26, ARF and LEA26+ARF as reference genes were further verified by analyzing the expression levels of csaCYCD3;1, csaRUL, cmoRUL, and cmoPIN in the graft union at different time points after grafting. Discussion This work is the first report of appropriate reference genes in grafted cucumber plants and provides useful information for the study of gene expression and molecular mechanisms in cucumber–pumpkin grafted plants.

Download Full-text