scholarly journals A novel medium throughput quantitative competitive PCR technology to simultaneously measure mRNA levels from multiple genes

2002 ◽  
Vol 30 (5) ◽  
pp. 20e-20 ◽  
Author(s):  
J. Zhang
Keyword(s):  
Mrna Levels ◽  
Competitive Pcr ◽  
Multiple Genes ◽  
Pcr Technology

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 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

Alternative splicing in plants

2008 ◽  
Vol 36 (3) ◽  
pp. 508-510 ◽  
Author(s):  
Craig G. Simpson ◽  
Dominika Lewandowska ◽  
John Fuller ◽  
Monika Maronova ◽  
Maria Kalyna ◽  
...  
Keyword(s):  
Gene Expression ◽  
Alternative Splicing ◽  
Reverse Transcription ◽  
Protein Function ◽  
Mrna Levels ◽  
Reverse Transcription Pcr ◽  
Multiple Genes ◽  
The Impact

The impact of AS (alternative splicing) is well-recognized in animal systems as a key regulator of gene expression and proteome complexity. In plants, AS is of growing importance as more genes are found to undergo AS, but relatively little is known about the factors regulating AS or the consequences of AS on mRNA levels and protein function. We have established an accurate and reproducible RT (reverse transcription)–PCR system to analyse AS in multiple genes. Initial studies have identified new AS events confirming that current values for the frequency of AS in plants are likely to be underestimates.

scholarly journals Apolipoprotein M inhibits proliferation and migration of larynx carcinoma cells

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Haixiang Xue ◽  
Miaomei Yu ◽  
Ying Zhou ◽  
Jun Zhang ◽  
Qinfeng Mu ◽  
...  
Keyword(s):  
Western Blot ◽  
Mrna Level ◽  
Carcinoma Cells ◽  
Mrna Levels ◽  
Apolipoprotein M ◽  
Protein Levels ◽  
Pcr Technology ◽  
And Migration ◽  
Cancer Tissues ◽  
Proliferation And Migration

Abstract Prior studies have shown that apolipoprotein M (APOM) is involved in the development of some cancers. Here we investigated the effects of APOM on larynx cancer (LC). 20 patients with vocal cord polyps and 18 patients with LC were included in this study. The protein and mRNA levels of the samples were analysed using the Wes-ProteinSimple system (or traditional Western blot) and PCR technology, respectively. APOM protein level in cancer tissues was lower than that in paracarcinomatous (P = 0.0003) and polyp tissues (P < 0.0001). APOM overexpression significantly inhibited TU686 cell proliferation (P < 0.0001) and migration (P < 0.01), and increased expression of vitamin D receptor (VDR, P < 0.0001) as well as nuclear factor erythroid 2-like 3 (NFE2L3, P = 0.0215). In addition, matrix metalloproteinase-10 (MMP-10) mRNA level was significantly reduced in the APOM overexpression group (P = 0.0077). However, Western blot analysis showed that APOM overexpression did not change VDR, NFE2L3 and MMP-10 protein levels (P > 0.05). In summary, APOM inhibits the proliferation and migration of LC cells, but may not be related to VDR, NFE2L3 and MMP-10, which needs further study.

scholarly journals IL-1α, IL-1β, IL-6, and TNF-α Steady-State mRNA Levels Analyzed by Reverse Transcription-Competitive PCR in Bone Marrow of Gonadectomized Mice

1998 ◽  
Vol 13 (2) ◽  
pp. 185-194 ◽  
Author(s):  
Rutger L. Van Bezooijen ◽  
Hetty C. M. Farih-Sips ◽  
Socrates E. Papapoulos ◽  
Clemens W. G. M. Löwik
Keyword(s):  
Bone Marrow ◽  
Steady State ◽  
Reverse Transcription ◽  
Mrna Levels ◽  
Competitive Pcr ◽  
Tnf Α

scholarly journals RNaseH-mediated simultaneous piggyback knockdown of multiple genes in adult zebrafish

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Revathi Raman ◽  
Mia Ryon ◽  
Pudur Jagadeeswaran
Keyword(s):  
Somatic Cells ◽  
Mrna Degradation ◽  
Whole Genome ◽  
Mrna Levels ◽  
Adult Zebrafish ◽  
Multiple Gene ◽  
Multiple Genes ◽  
Genome Wide ◽  
Multifactorial Disorders ◽  
Six Genes

AbstractWe recently developed a piggyback knockdown method that was used to knockdown genes in adult zebrafish. In this method, a vivo morpholino (VMO) piggybacks an antisense deoxyoligonucleotide (dO) into the somatic cells and reduces the cognate mRNA levels. In this paper, we tested whether we can piggyback more than one dO with one VMO. We designed various hybrids that had more than one dO that could be piggybacked with one VMO. We chose f7, f8, and αIIb genes and tested their knockdown by the appropriate assays. The knockdown with piggybacking either two or three dOs by one VMO yielded > 85% knockdown efficiency. We also performed knockdown of argonautes and rnaseh separately along with f7. We found the knockdown of f7 occurs when knockdown of argonautes happens and not when rnaseh knockdown was performed, suggesting that RNaseH is involved in mRNA degradation. In conclusion, we developed a method where we could knockdown three genes at one time, and by increasing the concentration of VMO by twofold, we could knockdown six genes simultaneously. These multiple gene knockdowns will not only increase the efficiency of the method in whole genome-wide knockdowns but will also be useful to study multifactorial disorders.

scholarly journals Levels of corticotrophin-releasing hormone receptor subtype 1 mRNA in pregnancy and during labour in human myometrium measured by quantitative competitive PCR

1998 ◽  
Vol 21 (2) ◽  
pp. 201-208 ◽  
Author(s):  
B Rodriguez-Linares ◽  
S Phaneuf ◽  
A Lopez Bernal ◽  
EA Linton
Keyword(s):  
Housekeeping Gene ◽  
Human Myometrium ◽  
Receptor Subtype ◽  
Mrna Levels ◽  
Competitive Pcr ◽  
Direct Role ◽  
Releasing Hormone ◽  
Tissue Changes ◽  
Pcr Method ◽  
In Pregnancy

It is suggested that corticotrophin-releasing hormone (CRH) is involved in parturition. We have previously reported the presence of the CRH receptor subtype 1 (CRH R1) in human uterine myocytes. The aim of the present study was to investigate whether expression of the CRH R1 in myometrial tissue changes in pregnancy and labour. We used a quantitative competitive PCR method to measure the mRNA levels of this receptor in non-pregnant and in term pregnant myometrium before and at different stages of labour. The levels of mRNA for the housekeeping gene for glucocerebrosidase (GCB) were also determined. The results were expressed as a ratio of CRH R1 and GCB mRNA levels. We have found that in pregnancy the CRH R1 is down-regulated from a ratio of 0.093+/-0.011 in non-pregnant myometrium to 0.012+/-0.005 (P<0.001) in term non-labouring myometrium. No significant changes were observed in the CRH R1:GCB ratio in tissues sampled within 13 h (0.013+/-0.004) from the start of labour. In summary, normalised levels of CRH R1 are down-regulated in pregnancy and do not change during labour. We speculate that our results do not support a direct role for the CRH R1 receptor in myometrial stimulation.

Quantification of eNOS mRNA in the canine cardiac vasculature by competitive PCR

2000 ◽  
Vol 278 (2) ◽  
pp. H658-H665 ◽  
Author(s):  
David Fulton ◽  
Andreas Papapetropoulos ◽  
Xiaoping Zhang ◽  
John D. Catravas ◽  
Thomas H. Hintze ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Coronary Artery ◽  
Left Ventricle ◽  
Molecular Mechanisms ◽  
Splenic Vein ◽  
Mrna Levels ◽  
Competitive Pcr ◽  
Aortic Endothelial Cells ◽  
Standard Curve ◽  
Enos Mrna

The goal of the present study was to develop a competitive PCR assay to measure changes in the expression of endothelial nitric oxide synthase (eNOS) mRNA levels throughout the canine vascular tree. A partial sequence of canine eNOS cDNA (1.86 kb), inducible NOS (1.95 kb), and neuronal NOS (1.16 kb) was cultured from canine aortic endothelial cells, LPS-treated canine splenic vein endothelial cells, and from canine left ventricle, respectively. Competitor eNOS cDNA (eNOS-C) was constructed via recombinant PCR. Thus, with the use of a standard curve competitive PCR with eNOS-C, the amount of eNOS mRNA in 500 ng of total RNA was greatest in the circumflex > right coronary artery > left anterior descending coronary artery > aorta. The isolation of coronary microvessels from the left ventricle was associated with an enrichment of endothelial cell markers such as eNOS, von Willebrand factor, and caveolin-1, an observation supported by the detection of up to 15-fold higher levels of eNOS mRNA in coronary microvessels relative to the larger arteries. The ability to quantify changes in eNOS mRNA levels throughout the canine vasculature should provide greater insight into the molecular mechanisms of how this gene is regulated in physiological and pathophysiological states.

ATRA Resolves the Differentiation Block in t(15;17) Myeloid Leukemia by Restoring PU.1 Expression.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 389-389 ◽  
Author(s):  
Beatrice U. Mueller ◽  
Thomas Pabst ◽  
Motomi Osato ◽  
Daniel G. Tenen
Keyword(s):  
Binding Activity ◽  
Mrna Levels ◽  
Gm Csf ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid ◽  
Translation Start ◽  
Conditional Expression ◽  
Mrna And Protein Expression ◽  
Pcr Technology ◽  
Differentiation Block

Abstract The transcription factor PU.1 is crucial for hematopoiesis. pu.1 knockout mice lack mature neutrophils, macrophages, B- and T-cells. PU.1 regulates important taget genes, like the M-CSF-, the G-CSF-, and the GM-CSF receptor promoter. Heterozygous mutations of the PU.1 gene have been found in some patients with AML, but not in AML with the t(15;17) encoding the PML-RARA fusion protein. These facts let us to hypothesize that decreased expression of PU.1 might contribute to the block in myeloid differentiation in t(15;17) AML. We therefore screened 141 AML patients for PU.1 mRNA expression using real-time PCR technology. We detected that t(15;17) AML patients (n=18) had significantly less PU.1 mRNA than AML-M2 patients (n=35), and 16fold less PU.1 mRNA than mature neutrophils. Moreover, the amount of PML-RARA transcripts in APL patients was inversely correlated to PU.1 mRNA levels. Conditional expression of PML-RARA in U937 cells suppressed PU.1 mRNA and PU.1 protein. HT93A cells derived from a t(15;17) AML-M3 patient nearly lack PU.1 mRNA and protein expression, and binding activity to the PU.1 site in the M-CSF-Receptor promoter is suppressed. Treatment of HT93A cells with all-trans retinoic acid (ATRA) powerfully restores PU.1 mRNA and protein and induces neutrophilic differentiation. We identified a region of extending −122 bp upstream of the translation start site in the PU.1 promoter mediates ATRA-induced activation of the PU.1 promoter in HT93A cells. Finally, conditional expression of PU.1 in HT93A cells is sufficient to trigger neutrophilic differentiation. This is the first report demonstrating that PU.1 is a critical target of the PML-RARA protein and an early ATRA-responsive gene.

Sign in / Sign up

Export Citation Format

Share Document