scholarly journals Reproducibility in the Quantification of mRNA Levels by RT-PCR-ELISA and RT Competitive-PCR-ELISA

BioTechniques ◽  
1998 ◽  
Vol 24 (4) ◽  
pp. 652-658 ◽  
Author(s):  
L.L. Hall ◽  
G.R. Bicknell ◽  
L. Primrose ◽  
J.H. Pringle ◽  
J.A. Shaw ◽  
...  
Keyword(s):  
Mrna Levels ◽  
Competitive Pcr ◽  
Rt Pcr

Negative Correlation Between Serum Levels of Homocysteine and Apolipoprotein M

2019 ◽  
Vol 19 (2) ◽  
pp. 120-126
Author(s):  
J. Wei ◽  
Y. Yu ◽  
Y. Feng ◽  
J. Zhang ◽  
Q. Jiang ◽  
...  
Keyword(s):  
Negative Correlation ◽  
Hepg2 Cells ◽  
Serum Levels ◽  
Significant Negative Correlation ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Apolipoprotein M ◽  
Protein Mass ◽  
Protein Levels ◽  
Phosphoinositide 3 Kinase

Background: Homocysteine (Hcy) has been suggested as an independent risk factor for atherosclerosis. Apolipoprotein M (apoM) is a constituent of the HDL particles. The goal of this study was to examine the serum levels of homocysteine and apoM and to determine whether homocysteine influences apoM synthesis. Methods: Serum levels of apoM and Hcy in 17 hyperhomocysteinemia (HHcy) patients and 19 controls were measured and their correlations were analyzed. Different concentrations of homocysteine (Hcy) and LY294002, a specific phosphoinositide 3- kinase (PI3K) inhibitor, were used to treat HepG2 cells. The mRNA levels were determined by RT-PCR and the apoM protein mass was measured by western blot. Results: We found that decreased serum apoM levels corresponded with serum HDL levels in HHcy patients, while the serum apoM levels showed a statistically significant negative correlation with the serum Hcy levels. Moreover, apoM mRNA and protein levels were significantly decreased after the administration of Hcy in HepG2 cells, and this effect could be abolished by addition of LY294002. Conclusions: resent study demonstrates that Hcy downregulates the expression of apoM by mechanisms involving the PI3K signal pathway.

scholarly journals Phenotypical and Functional Characteristics of Human Regulatory T Cells during Ex Vivo Maturation from CD4+ T Lymphocytes

2021 ◽  
Vol 11 (13) ◽  
pp. 5776
Author(s):  
Varvara G. Blinova ◽  
Natalia S. Novachly ◽  
Sofya N. Gippius ◽  
Abdullah Hilal ◽  
Yulia A. Gladilina ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Ex Vivo ◽  
Pcr Analysis ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Inflammatory Reactions ◽  
Effector Cells ◽  
Cycle Regulation ◽  
Further Development

Regulatory T cells (Tregs) participate in the negative regulation of inflammatory reactions by suppressing effector cells. In a number of autoimmune disorders, the suppressive function and/or the number of Tregs is compromised. The lack of active functioning Tregs can be restored with adoptive transfer of expanded ex vivo autologous Tregs. In our study, we traced the differentiation and maturation of Tregs CD4+CD25+FoxP3+CD127low over 7 days of cultivation from initial CD4+ T cells under ex vivo conditions. The resulting ex vivo expanded cell population (eTregs) demonstrated the immune profile of Tregs with an increased capacity to suppress the proliferation of target effector cells. The expression of the FoxP3 gene was upregulated within the time of expansion and was associated with gradual demethylation in the promotor region of the T cell-specific demethylation region. Real-time RT-PCR analysis revealed changes in the expression profile of genes involved in cell cycle regulation. In addition to FOXP3, the cells displayed elevated mRNA levels of Ikaros zinc finger transcription factors and the main telomerase catalytic subunit hTERT. Alternative splicing of FoxP3, hTERT and IKZF family members was demonstrated to be involved in eTreg maturation. Our data indicate that expanded ex vivo eTregs develop a Treg-specific phenotype and functional suppressive activity. We suggest that eTregs are not just expanded but transformed cells with enhanced capacities of immune suppression. Our findings may influence further development of cell immunosuppressive therapy based on regulatory T cells.

scholarly journals The usefulness of competitive PCR: airway gene expression of IL-5, IL-4, IL-4δ2, IL-2, and IFNγ in asthma

Thorax ◽  
2001 ◽  
Vol 56 (7) ◽  
pp. 541-548
Author(s):  
E M Glare ◽  
M Divjak ◽  
M J Bailey ◽  
E H Walters
Keyword(s):  
Gene Expression ◽  
Inhaled Corticosteroids ◽  
In Situ Hybridisation ◽  
Housekeeping Gene ◽  
Cross Sectional Study ◽  
Mrna Levels ◽  
Competitive Pcr ◽  
Cross Sectional ◽  
Steroid Use ◽  
Biopsy Specimens

BACKGROUNDAsthma has been described as an eosinophilic bronchitis driven by interleukin(IL)-4 and IL-5. The quantification of cytokine mRNA levels in airway samples has been confounded by housekeeping gene expression which differs between and within asthmatics and controls.METHODSThe usefulness of competitive reverse transcriptase-polymerase chain reaction (RT-PCR) that is independent of housekeeping gene expression for quantitating the mRNA for interferon (IFN)γ, IL-2, IL-5, IL-4 and its receptor antagonist encoding splicing variant IL-4δ2 was determined in a cross sectional study of 45 normal control subjects and 111 with asthma.RESULTSAtopic controls and atopic asthmatic subjects expressed more IL-5 than non-atopic controls (p<0.02) in bronchoalveolar lavage (BAL) cells, but not in biopsy specimens. IL-5 mRNA expression in BAL cells from asthmatic subjects using inhaled corticosteroids (ICS) was significantly lower than those not receiving ICS (p=0.04). IL-2 mRNA levels differed with steroid use in biopsy specimens but not in BAL cells. IFNγ, IL-4, and IL-4δ2 mRNA levels did not differ between any groups and were not affected by steroid use. IL-4 and IL-4δ2 mRNA levels were positively correlated (p<0.0001), suggesting coordinated transcription.CONCLUSIONSWhile the signal differentiation of competitive PCR in asthma may rival that of in situ hybridisation and immunohistochemistry, the method is expensive and wasteful of material.

scholarly journals Quantitative assessment of CYP11B1 and CYP11B2 expression in aldosterone-producing adenomas

2002 ◽  
pp. 795-802 ◽  
Author(s):  
F Fallo ◽  
V Pezzi ◽  
L Barzon ◽  
P Mulatero ◽  
F Veglio ◽  
...  
Keyword(s):  
Real Time ◽  
Secretory Activity ◽  
Zona Glomerulosa ◽  
Zona Fasciculata ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Pathophysiological Role ◽  
Aldosterone Production

BACKGROUND: The presence and pathophysiological role of CYP11B1 (11beta-hydroxylase) gene in the zona glomerulosa of human adrenal cortex is still controversial. METHODS: In order to specifically quantify CYP11B1, CYP11B2 (aldosterone synthase) and CYP17(17alpha-hydroxylase) mRNA levels, we developed a real-time RT-PCR assay and examined the expression in a series of adrenal tIssues, including six normal adrenals from patients adrenalectomized for renal cancer and twelve aldosterone-producing adenomas (APA) from patients with primary aldosteronism. RESULTS: CYP11B1 mRNA levels were clearly detected in normal adrenals, which comprised both zona glomerulosa and fasciculata/reticularis cells, but were also measured at a lower range (P<0.05) in APA. The levels of CYP11B2 mRNA were lower (P<0.005) in normal adrenals than in APA. CYP17 mRNAlevels were similar in normal adrenals and in APA. In patients with APA, CYP11B2 and CYP11B1 mRNA levels were not correlated either with basal aldosterone or with the change from basal aldosterone in response to posture or to dexamethasone. No correlation between CYP11B1 mRNA or CYP11B2 mRNA and the percentage of zona fasciculata-like cells was observed in APA. CONCLUSIONS: Real-time RT-PCR can be reliably used to quantify CYP11B1 and CYP11B2 mRNA levels in adrenal tIssues. Expression of CYP11B1 in hyperfunctioning zona glomerulosa suggests an additional formation of corticosterone via 11beta-hydroxylase, providing further substrate for aldosterone biosynthesis. CYP11B1 and CYP11B2 mRNA levels in APA are not related to the in vivo secretory activity of glomerulosa cells, where post-transcriptional factors might ultimately regulate aldosterone production.

Relative Levels of mRNA Encoding Enamel Proteins in Enamel Organ Epithelia and Odontoblasts

2003 ◽  
Vol 82 (12) ◽  
pp. 982-986 ◽  
Author(s):  
T. Nagano ◽  
S. Oida ◽  
H. Ando ◽  
K. Gomi ◽  
T. Arai ◽  
...  
Keyword(s):  
Tooth Enamel ◽  
Structural Proteins ◽  
Enamel Organ ◽  
Maturation Stage ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Enamel Protein ◽  
Enamel Proteins ◽  
Enamel Layer

Amelogenin, enamelin, sheathlin (ameloblastin/ amelin), enamelysin (MMP-20), and KLK4 (EMSP-1) are the major structural proteins and proteinases in developing tooth enamel. Recently, odontoblasts were reported to express amelogenin, the most abundant enamel protein. In this study, we hypothesized that odontoblasts express all enamel proteins and proteases, and we measured their relative mRNA levels in enamel organ epithelia and odontoblasts associated with porcine secretory- and maturation-stage enamel by RT-PCR, using a LightCycler instrument. The results showed that amelogenin mRNA in secretory-stage EOE is 320-fold higher than in odontoblasts beneath secretory-stage enamel, and over 20,000-fold higher than in odontoblasts under maturation-stage enamel. Similar results were obtained for enamelin and sheathlin. Enamelysin mRNA levels were equivalent in these two tissues, while KLK4 mRNA was higher in odontoblasts than in secretory-stage EOE. These results support the conclusion that odontoblasts are involved in the formation of the enamel layer adjacent to enamel-dentin junction.

scholarly journals Steroidogenic enzymes mRNA expression profile and steroids production in bovine theca cells cultured in vitro and stimulated with angiotensin II

2015 ◽  
Vol 45 (4) ◽  
pp. 704-710 ◽  
Author(s):  
Melânia Lazzari Rigo ◽  
Andressa Minussi Pereira Dau ◽  
Werner Giehl Glanzner ◽  
Manoel Martins ◽  
Renato Zanella ◽  
...  
Keyword(s):  
High Performance ◽  
Culture Media ◽  
Mrna Levels ◽  
Ang Ii ◽  
Rt Pcr ◽  
Steroidogenic Enzymes ◽  
Theca Cells ◽  
Mrna Profile ◽  
Dose Response Effect

The main objective of this study was to detect the steroidogenic effects of Ang II in bovine theca cells in vitro. Bovine theca cells were obtained from follicles (larger than 10mm of diameter) collected from a local abattoir and submitted to different treatments in a sequence of experiments. In experiment 1, CYP17A1 mRNA profile was evaluated in LH- (10ng ml-1) and Ang II-treated (0.1µM) theca cells. In experiment 2, a dose-response effect of Ang II (0.001; 0.1 e 10µM) plus insulin (100ng ml-1) and LH (100ng ml-1) was evaluated on steroidogenesis of bovine theca cells. Experiment 3 explored the effects of saralasin (an antagonist of Ang II receptors) on steroid production and steroidogenic enzymes regulation in theca cells. After 24 hours, culture media from experiments 2 and 3 was collected to evaluate testosterone and androstenedione levels by High-Performance Liquid Chromatography. In parallel, mRNA levels of key steroidogenic enzymes (HSD3B2, CYP11A1, CYP17A1) and STAR were assessed by RT-PCR. There was no difference in testosterone and androstenedione production between treated and controls groups, as well as in mRNA levels of the evaluated genes. In conclusion, the results suggest that Ang II does not regulate steroidogenesis in bovine theca cells

scholarly journals Exercise-induced changes in β-adrenergic-receptor mRNA level measured by competitive RT-PCR

1997 ◽  
Vol 82 (6) ◽  
pp. 1926-1931 ◽  
Author(s):  
Nobuharu Fujii ◽  
Takeshi Shibata ◽  
Sachiko Homma ◽  
Haruo Ikegami ◽  
Kazuo Murakami ◽  
...  
Keyword(s):  
Adrenergic Receptor ◽  
Human Lymphocytes ◽  
Mrna Level ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Receptor Mrna ◽  
Exercise Induced ◽  
Induced Changes ◽  
Receptor Mrna Level

Fujii, Nobuharu, Takeshi Shibata, Sachiko Homma, Haruo Ikegami, Kazuo Murakami, and Hitoshi Miyazaki. Exercise-induced changes in β-adrenergic-receptor mRNA level measured by competitive RT-PCR. J. Appl. Physiol. 82(6): 1926–1931, 1997.—Competitive reverse transcription-polymerase chain reaction (RT-PCR) analysis was used to clarify whether dynamic exercise-induced increases in β-adrenergic-receptor (β-AR) number in human lymphocytes are accompanied by increases in the β-AR mRNA level. Sixteen healthy subjects performed cycle ergometry until exhaustion. Before and immediately after exercise, peripheral blood was drawn from a forearm vein for preparation of lymphocytes. Both the β-AR mRNA level and the β-AR number were significantly increased by exercise. The changes in β-AR mRNA level and β-AR number were significantly correlated ( r = 0.63, P < 0.01). This finding suggests that a rapid increase in β-AR mRNA level might be an early adaptive response of the sympathetic nervous system to dynamic exercise. In vitro incubation of lymphocytes with epinephrine had no effect on β-AR mRNA levels, nor did adenosine 3′,5′-cyclic monophosphate, protein kinase C, or intracellular Ca2+ increase the β-AR mRNA level in vitro. Therefore, it appears that other mechanisms underlie the exercise-induced elevation of β-AR mRNA levels in human lymphocytes.

Temporary increased cytokine mRNA levels in bone marrow after ovariectomy; using competitive RT-PCR

Bone ◽  
1995 ◽  
Vol 17 (6) ◽  
pp. 587
Author(s):  
R.L. van Bezooiien ◽  
S.E. Papapoulos ◽  
C.W.G.M. Löwik
Keyword(s):  
Bone Marrow ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Cytokine Mrna
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