scholarly journals Quantitative Gene Expression Assays without Target Amplification

2003 ◽  
Vol 8 (2) ◽  
pp. 30-33 ◽  
Author(s):  
Deirdre Trainor ◽  
James Tolley ◽  
Carl LaCerte ◽  
Barbara Gaynor ◽  
Toddy Sewell ◽  
...  
Keyword(s):  
Gene Expression ◽  
Signal Amplification ◽  
Mononuclear Cells ◽  
Primary Cultures ◽  
Cdna Clones ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Assay Sensitivity ◽  
Mrna Targets ◽  
Mrna Induction

Evaluating changes in gene expression is essential when identifying and validating new drug targets, however, the methods used to measure transcription are laborious, time-consuming and expensive ( e.g., RT-PCR, microarray or northern blot). High Performance Signal Amplification (HPSA™) gene expression assays quantitate particular mRNA targets directly in cell lysate samples using DNA probe hybridization and fluorescent signal amplification. The assay format eliminates the need for RNA purification prior to testing and does not require RT-PCR amplification. The HPSA™ protocol involves three steps carried out at 37°C in 96- or 384-well plates, making the technique amenable to automation. Cellular mRNA levels are quantitated relative to a standard curve consisting of purified in vitro RNA transcripts derived from cDNA clones. Assay sensitivity is in the low attomole range and can detect mRNA expressed at twenty copies per cell. A number of HPSA™ assays have been developed for cytokine, housekeeping and cytochrome P450 messages. Gene induction profiles were monitored in cell lines or peripheral blood mononuclear cells (PBMC) treated with activators such as phorbol 12-myristate-13-acetate (PMA), ionomycin or phytohemagglutinin (PHA). Recent validation studies demonstrated IL-9 mRNA induction in primary cultures of T-cells treated with PMA and anti-CD3. Similar testing with PBMCs showed significant IL-13 mRNA induction after 48 hours of treatment with PMA, thymosin and staphylococcal enterotoxin. Cytokine STATE® panels are being constructed according to functional categories such as innate or adaptive immunity to better characterize changes in transcription patterns during an immune response. The HPSA™ gene expression assays offer a rapid and convenient alternative to more cumbersome, expensive methods.

scholarly journals Human mononuclear cells express 12-LX: coordinated mRNA regulation with 5-LX and FLAP genes

Blood ◽  
1996 ◽  
Vol 87 (1) ◽  
pp. 331-340
Author(s):  
WE Kaminski ◽  
E Jendraschak ◽  
K Baumann ◽  
R Kiefl ◽  
S Fischer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mrna Expression ◽  
Time Course ◽  
Cell Activation ◽  
Mononuclear Cells ◽  
Constitutive Expression ◽  
Ionophore A23187 ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Human Mononuclear Cells

Lipoxygenases (LXs) catalyze formation of leukotrienes and hydroxy-eicosatetraenoic acids (HETEs), proinflammatory, and spasmogenic autacoids that are critical for host defense systems. We studied the expression and regulation of LX genes (12-LX, 5-LX, and 15-LX) and the 5-lipoxygenase activating protein (FLAP) in human mononuclear cells (MNC) and granulocytes using a quantitative reverse transcription polymerase chain reaction (RT-PCR) technique. We show that 12-LX mRNA is constitutively expressed in resting platelet-free MNC. 12-LX gene expression was upregulated by activation with lipopolysaccharide (LPS). The formation of 12-HETE was inducible with ionophore in MNC, as assessed by high-performance liquid chromatography (HPLC) and gas chromatography, and increased after LPS pretreatment. In addition to 12- LX, resting MNC expressed the genes for 5-LX and FLAP constitutively. Quantitative time course analyses of 12-LX, 5-LX, and FLAP gene expression suggested coregulation of 12-LX and FLAP mRNAs, and reciprocal regulation of 5-LX and FLAP mRNAs. During cell stimulation with LPS 5-LX mRNA levels remained unchanged, whereas FLAP gene expression increased. No 15-LX mRNA expression or 15-HETE formation was detectable in unstimulated and activated MNC. In contrast to MNC, quantitative RT-PCR mRNA analysis showed intermittent intraindividual expression of the 5-LX and FLAP genes in resting granulocytes. mRNAs for 12-LX and 15-LX were not expressed. On stimulation of granulocytes ex vivo, mRNA expression of 5-LX and FLAP was upregulated. Stimulation by LPS differed from that by ionophore A23187. Neither LPS nor ionophore induced gene expression of 12-LX or 15-LX in granulocytes. Our data indicate that resting human MNC and granulocytes express LX and FLAP genes in a cell-specific manner. Cell activation induces coordinated upregulation of 12-LX and FLAP genes in MNC, and 5-LX and FLAP genes in granulocytes, respectively. The constitutive expression of 12-LX mRNA, its upregulation on cell activation, and the formation of 12-HETE clearly indicate the presence of a functional 12-LX in human MNC.

scholarly journals Human mononuclear cells express 12-LX: coordinated mRNA regulation with 5-LX and FLAP genes

Blood ◽  
1996 ◽  
Vol 87 (1) ◽  
pp. 331-340 ◽  
Author(s):  
WE Kaminski ◽  
E Jendraschak ◽  
K Baumann ◽  
R Kiefl ◽  
S Fischer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mrna Expression ◽  
Time Course ◽  
Cell Activation ◽  
Mononuclear Cells ◽  
Constitutive Expression ◽  
Ionophore A23187 ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Human Mononuclear Cells

Abstract Lipoxygenases (LXs) catalyze formation of leukotrienes and hydroxy-eicosatetraenoic acids (HETEs), proinflammatory, and spasmogenic autacoids that are critical for host defense systems. We studied the expression and regulation of LX genes (12-LX, 5-LX, and 15-LX) and the 5-lipoxygenase activating protein (FLAP) in human mononuclear cells (MNC) and granulocytes using a quantitative reverse transcription polymerase chain reaction (RT-PCR) technique. We show that 12-LX mRNA is constitutively expressed in resting platelet-free MNC. 12-LX gene expression was upregulated by activation with lipopolysaccharide (LPS). The formation of 12-HETE was inducible with ionophore in MNC, as assessed by high-performance liquid chromatography (HPLC) and gas chromatography, and increased after LPS pretreatment. In addition to 12- LX, resting MNC expressed the genes for 5-LX and FLAP constitutively. Quantitative time course analyses of 12-LX, 5-LX, and FLAP gene expression suggested coregulation of 12-LX and FLAP mRNAs, and reciprocal regulation of 5-LX and FLAP mRNAs. During cell stimulation with LPS 5-LX mRNA levels remained unchanged, whereas FLAP gene expression increased. No 15-LX mRNA expression or 15-HETE formation was detectable in unstimulated and activated MNC. In contrast to MNC, quantitative RT-PCR mRNA analysis showed intermittent intraindividual expression of the 5-LX and FLAP genes in resting granulocytes. mRNAs for 12-LX and 15-LX were not expressed. On stimulation of granulocytes ex vivo, mRNA expression of 5-LX and FLAP was upregulated. Stimulation by LPS differed from that by ionophore A23187. Neither LPS nor ionophore induced gene expression of 12-LX or 15-LX in granulocytes. Our data indicate that resting human MNC and granulocytes express LX and FLAP genes in a cell-specific manner. Cell activation induces coordinated upregulation of 12-LX and FLAP genes in MNC, and 5-LX and FLAP genes in granulocytes, respectively. The constitutive expression of 12-LX mRNA, its upregulation on cell activation, and the formation of 12-HETE clearly indicate the presence of a functional 12-LX in human MNC.

scholarly journals Simultaneous Fluorescent Detection of Two mRNA Targets by High Performance Signal Amplification

2000 ◽  
Vol 5 (1) ◽  
pp. 44-44
Author(s):  
Frank R. Gonzales
Keyword(s):  
Gene Expression ◽  
Signal Amplification ◽  
High Performance ◽  
Dna Probe ◽  
Fluorescent Detection ◽  
Quantum Yields ◽  
Target System ◽  
Mrna Targets ◽  
Mrna Induction ◽  
Actin Mrna

Development of drug candidates, which modulate cytokine responses or metabolic pathways by targeting gene expression, requires analytical systems that measure specific messenger RNAs rapidly and accurately and that can be adapted to high throughput screening operations. Chromagen's High Performance Signal Amplification (HPSA), system, a DNA probe hybridization method, has been enhanced to measure two different mRNA targets simultaneously in the same micro-plate well. Multi-target testing using HPSA takes advantage of a family of low molecular weight fluorescent dyes with large quantum yields, which exhibit distinct excitation and emission wavelengths. A two-target model system using cultured human THP-1 cells examined IL-1β mRNA induction in response to bacterial lipopolysaccharide (LPS) exposure and also monitored intrinsic β-actin mRNA as a housekeeping gene transcript. An oligonucleotide DNA probe complementary to IL-1β RNA was labeled with a reporter ligand while a β-actin DNA probe was linked directly to an enzyme. Sample mRNA was captured onto the surface of micro-plate wells and hybridized to both DNA probes. After removing non-hybridized probe, a second enzyme conjugate that specifically recognizes the IL-1β DNA probe reporter ligand was allowed to bind. The two enzyme systems were distinguished by using substrates labeled with different fluorescent tags (one for IL-1β and the other for β-actin). Resolution of the two fluorescent products was carried out with Chromagen's high-sensitivity, photon-counting fluorometer. This system was capable of detecting synthetic RNA targets for IL-1β and β-actin in the attomole range. Authentic β-actin mRNA was measured in 50,000 uninduced cells and this signal could be specifically competed away by addition of excess unlabeled β-actin probe during hybridization. A time course of IL-1β mRNA induction in THP-1 cells by LPS revealed that peak induction occurred after 2–3 hours. The β-actin mRNA level showed an initial decrease, but remained relatively constant throughout the remaining time points. Results obtained with the dual detection format paralleled those generated when each target was measured separately in its single-target gene expression assay. Chromagen's HPSA two-target system not only augments screening information, but also saves testing time and conserves reagents. The potential of this system is being explored with different gene targets of therapeutic value and other housekeeping genes.

Cloning of rabbit prolactin cDNA and prolactin gene expression in the rabbit mammary gland

1996 ◽  
Vol 16 (1) ◽  
pp. 27-37 ◽  
Author(s):  
L Gabou ◽  
M Boisnard ◽  
I Gourdou ◽  
H Jammes ◽  
J-P Dulor ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mammary Gland ◽  
Amino Acid ◽  
Untranslated Regions ◽  
Pcr Analysis ◽  
Cdna Clones ◽  
Rt Pcr ◽  
Prolactin Gene ◽  
New Zealand White Rabbits ◽  
Rna Probe

ABSTRACT cDNA clones coding for rabbit prolactin were isolated from a pituitary library using a rat prolactin RNA probe. One cDNA contained 873 bases including the entire coding sequence of rabbit prolactin, its signal peptide and the 5′ and 3′ untranslated regions of 44 and 145 nucleotides respectively. The deduced amino acid sequence of the cloned prolactin cDNA presented a 93–78% identity with mink, porcine and human prolactins. The prolactin gene transcription was investigated by RT-PCR analysis in several organs of midlactating New Zealand White rabbits. The ectopic transcription of the prolactin gene was examined in more detail in the mammary gland. A strong PCR signal was detected in the mammary gland of virgin does and was also observed during pregnancy and at the beginning of lactation. This PCR signal was very weak in mid-lactating and absent in post-weaning mammary gland.

scholarly journals Gene Expression of Adhesion Molecules in Endothelial Cells from Patients with Peripheral Arterial Disease Is Reduced after Surgical Revascularization and Pharmacological Treatment

2013 ◽  
Vol 2013 ◽  
pp. 1-8 ◽  
Author(s):  
Franca Marino ◽  
Luigina Guasti ◽  
Matteo Tozzi ◽  
Laura Schembri ◽  
Luana Castiglioni ◽  
...  
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
Mrna Expression ◽  
Adhesion Molecules ◽  
Mononuclear Cells ◽  
Venous Blood ◽  
Statin Treatment ◽  
Early Event ◽  
Mrna Levels ◽  
Peripheral Blood Mononuclear

Atherosclerosis is an inflammatory disease characterized by immunological activity, in which endothelial dysfunction represents an early event leading to subsequent inflammatory vascular damage. We investigated gene expression of the adhesion molecules (AMs) ICAM-1, VCAM-1, andβ1-integrin in endothelial cells (ECs) isolated from venous blood (circulating EC, cEC) and purified from femoral plaques (pEC) obtained from 9 patients with peripheral artery disease (PAD) submitted to femoral artery thrombendarterectomy (FEA). In addition, in peripheral blood mononuclear cells (PBMCs) of the same subjects, we investigated gene expression of IFN-γ, IL-4, TGF-β, and IL-10. Patients were longitudinally evaluated 1 month before surgery, when statin treatment was established, at the time of surgery, and after 2 and 5 months. All AM mRNA levels, measured by means of real-time PCR, in cEC diminished during the study, up to 41–50% of initial levels at followup. AM mRNA expression was significantly higher in pEC than in cEC. During the study, in PBMCs, TGF-βand IL-10 mRNA levels remained unchanged while IFN-γand IL-4 levels increased; however, the ratio IFN-γ/IL-4 showed no significant modification. In PAD patients, FEA and statin treatment induce a profound reduction of AM expression in cEC and affect cytokine mRNA expression in PBMCs.

scholarly journals Monitoring gene expression changes in bovine oviduct epithelial cells during the oestrous cycle

2004 ◽  
Vol 32 (2) ◽  
pp. 449-466 ◽  
Author(s):  
S Bauersachs ◽  
S Rehfeld ◽  
SE Ulbrich ◽  
S Mallok ◽  
K Prelle ◽  
...  
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Expression Profiles ◽  
Cdna Array ◽  
Oestrous Cycle ◽  
Cdna Clones ◽  
Rt Pcr ◽  
Expression Levels ◽  
Starting Point ◽  
Oviduct Epithelial Cells

The oviduct epithelium undergoes marked morphological and functional changes during the oestrous cycle. To study these changes at the level of the transcriptome we did a systematic gene expression analysis of bovine oviduct epithelial cells at oestrus and dioestrus using a combination of subtracted cDNA libraries and cDNA array hybridisation. A total of 3072 cDNA clones of two subtracted libraries were analysed by array hybridisation with cDNA probes derived from six cyclic heifers, three of them slaughtered at oestrus and three at dioestrus. Sequencing of cDNAs showing significant differences in their expression levels revealed 77 different cDNAs. Thirty-seven were expressed at a higher level at oestrus, for the other 40 genes expression levels were higher at dioestrus. The identified genes represented a variety of functional classes. During oestrus especially genes involved in the regulation of protein secretion and protein modification, and mRNAs of secreted proteins, were up-regulated, whereas during dioestrus particularly transcripts of genes involved in transcription regulation showed a slight up-regulation. The concentrations of seven selected transcripts were quantified by real-time RT-PCR to validate the cDNA array hybridisation data. For all seven transcripts, RT-PCR results were in excellent correlation (r>0.92) with the results obtained by array hybridisation. Our study is the first to analyse changes in gene expression profiles of bovine oviduct epithelial cells during different stages of the oestrous cycle, providing a starting point for the clarification of the key transcriptome changes in these cells.

scholarly journals Regulation of c-jun gene expression in human T lymphocytes

Blood ◽  
1993 ◽  
Vol 81 (6) ◽  
pp. 1540-1548 ◽  
Author(s):  
D Chauhan ◽  
SM Kharbanda ◽  
E Rubin ◽  
BA Barut ◽  
A Mohrbacher ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Messenger Rna ◽  
Jurkat Cells ◽  
Pokeweed Mitogen ◽  
Mrna Levels ◽  
Dependent Manner ◽  
B Cell Differentiation ◽  
Normal Peripheral Blood ◽  
Mrna Induction

Abstract The present studies have examined the effects of mitogens on induction of early response gene expression in normal peripheral blood T and Jurkat cells. Pokeweed mitogen (PWM) or anti-CD3 significantly increases c-jun messenger RNA (mRNA) levels in T cells. This transient PWM-related increase in c-jun transcripts is maximal after 15 to 30 minutes of exposure of T cells to PWM. PWM induces c-jun gene expression in a concentration-dependent manner. Moreover, PWM similarly induces expression of other genes coding for leucine zipper transcription factors, ie, jun-B and c-fos. Nuclear run on assays demonstrate that PWM treatment is associated with an increased rate of c-jun gene transcription. Transient expression assays with c-jun promoter fragments linked to the chloramphenicol acetyltransferase gene suggest that the PWM-induced increase in transcription is mediated by the AP-1 transcription factor complex. Moreover, treatment of T cells with actinomycin D to block further transcription before their culture with PWM suggests that the increase in c-jun gene expression by PWM is also regulated at least in part by a posttranscriptional mechanism. Cycloheximide does not alter c-jun mRNA induction by PWM. Finally, given that PWM induces B-cell differentiation in an interleukin-6 (IL- 6)-mediated, T-cell-dependent manner, the relationship of c-jun and IL- 6 gene expression in PWM-stimulated T cells was examined. The induction of IL-6 mRNA in T cells stimulated by PWM occurs after maximal induction of c-jun mRNA, at a time when the latter is no longer detectable. These findings suggest that PWM induces c-jun gene expression in T cells by a transcriptional and posttranscriptional mechanism and that AP-1 confers PWM inducibility of this gene. Because the IL-6 promoter has several potential transcriptional control elements, one of which is an AP-1-binding site, future experiments will evaluate the role of c-jun in the regulation of PWM-induced IL-6 synthesis by T cells.

Phorbol 12-myristate 13-acetate alters SR Ca(2+)-ATPase gene expression in cultured neonatal rat heart cells

1996 ◽  
Vol 271 (3) ◽  
pp. H1031-H1039 ◽  
Author(s):  
M. Qi ◽  
J. W. Bassani ◽  
D. M. Bers ◽  
A. M. Samarel
Keyword(s):  
Gene Expression ◽  
Sarcoplasmic Reticulum ◽  
Mrna Stability ◽  
Primary Cultures ◽  
Neonatal Rat ◽  
Response To Treatment ◽  
Similar Degree ◽  
Heart Cells ◽  
Mrna Levels ◽  
Atpase Gene

Primary cultures of neonatal rat ventricular myocytes were used to examine how the cardiac myocyte cytoplasmic Ca2+ ([Ca2+]i) transient and sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2) gene expression change in response to treatment with the protein kinase C activator phorbol 12-myristate 13-acetate (PMA). Exposure of neonatal myocytes to PMA (200 nM, 48-72 h) produced myocyte growth and a 70% prolongation of the half-time for [Ca2+]i decline induced by potassium depolarization in the absence of extracellular Na+ (in which the sarcoplasmic reticulum Ca2+ pump is the main mechanism responsible for [Ca2+]i decline). The reduced rate of [Ca2+]i transient decline corresponded to a 53% reduction in SERCA2 protein levels and a 43% reduction in SERCA2 mRNA levels as compared with control myocytes. Exposure to PMA for as little as 30 min or for as long as 48 h produced a similar degree of SERCA2 mRNA downregulation over time. PMA-induced downregulation of SERCA2 mRNA levels was blocked by either 10 nM staurosporine or 4 microM chelerythrine, whereas treatment with either agent alone increased SERCA2 mRNA levels as compared with control cells. Actinomycin D mRNA stability assays revealed that PMA treatment appeared to markedly destabilize the relatively long-lived SERCA2 mRNA transcript. Taken together, these results indicate that downregulation of SERCA2 gene by PMA in cultured neonatal myocytes occurs at least in part by alterations in mRNA stability and results in functional alterations in [Ca2+]i decline that are similar to that observed in the hypertrophied and failing adult myocardium.

Acute hypoxia stimulates renin secretion and renin gene expression in vivo but not in vitro

1997 ◽  
Vol 272 (4) ◽  
pp. R1105-R1111 ◽  
Author(s):  
T. Ritthaler ◽  
K. Schricker ◽  
F. Kees ◽  
B. Kramer ◽  
A. Kurtz
Keyword(s):  
Gene Expression ◽  
Acute Hypoxia ◽  
Primary Cultures ◽  
Renin Secretion ◽  
Plasma Norepinephrine ◽  
Mrna Levels ◽  
Sprague Dawley ◽  
Renin Gene ◽  
Renin Mrna

This study aimed at examining the influence of acute hypoxia on renin secretion and renin gene expression in the kidney. To this end, male Sprague-Dawley rats were exposed to severe hypoxic stress (8% O2) or to carbon monoxide (0.1% CO) for 6 h, and plasma renin activity (PRA) and renal renin mRNA levels were determined. PRA values increased from 3 to 13 and 10 ng angiotensin I x h(-1) x ml(-1), and renin mRNA levels increased by 120 and 100% during hypoxia and CO, respectively. Lowering the PO2 from 150 to 20 or 7 mmHg in the gas atmosphere of primary cultures of renal juxtaglomerular cells had no influence on renin secretion and renin gene expression after 6 and 20 h. Our findings thus suggest that both arterial and venous hypoxia can be powerful stimulators of renin secretion and renin gene expression in vivo. Because renal denervation did not prevent stimulation of the renin system by hypoxia, the effect could be indirectly mediated via the baroreceptor-macula densa mechanism. Another potential mediator of the effect could be circulating catecholamines, since we found that plasma norepinephrine increased from 0.7 to 1.5 and 2.4 ng/ml and plasma epinephrine increased from 0.3 to 1.4 and 2.7 ng/ml during hypoxia and CO inhalation, respectively.

Gene expression in the rat supraoptic nucleus induced by chronic hyperosmolality versus hyposmolality

2000 ◽  
Vol 279 (4) ◽  
pp. R1239-R1250 ◽  
Author(s):  
Eric Glasgow ◽  
Takashi Murase ◽  
Bingjun Zhang ◽  
Joseph G. Verbalis ◽  
Harold Gainer
Keyword(s):  
Gene Expression ◽  
Supraoptic Nucleus ◽  
Cdna Libraries ◽  
Global Changes ◽  
Cdna Clones ◽  
Mrna Levels ◽  
Magnocellular Neurons ◽  
In Situ Hybridization Histochemistry ◽  
Functional Classes

Magnocellular neurons of the hypothalamo-neurohypophysial system play a fundamental role in the maintenance of body homeostasis by secreting vasopressin and oxytocin in response to systemic osmotic perturbations. During chronic hyperosmolality, vasopressin and oxytocin mRNA levels increase twofold, whereas, during chronic hyposmolality, these mRNA levels decrease to 10–20% of that of normoosmolar control animals. To determine what other genes respond to these osmotic perturbations, we have analyzed gene expression during chronic hyper- versus hyponatremia. Thirty-seven cDNA clones were isolated by differentially screening cDNA libraries that were generated from supraoptic nucleus tissue punches from hyper- or hyponatremic rats. Further analysis of 12 of these cDNAs by in situ hybridization histochemistry confirmed that they are osmotically regulated. These cDNAs represent a variety of functional classes and include cytochrome oxidase, tubulin, Na+-K+-ATPase, spectrin, PEP-19, calmodulin, GTPase, DnaJ-like, clathrin-associated, synaptic glycoprotein, regulator of GTPase stimulation, and gene for oligodendrocyte lineage-myelin basic proteins. This analysis therefore suggests that adaptation to chronic osmotic stress results in global changes in gene expression in the magnocellular neurons of the supraoptic nucleus.

Sign in / Sign up

Export Citation Format

Share Document