Quantitation of gene expression in neural precursors by reverse-transcription polymerase chain reaction using self-quenched, fluorogenic primers

Lowe, Brian; Avila, Herbert A.; Bloom, Fredric R.; Gleeson, Martin A.; Kusser, Wolfgang

doi:10.1016/s0003-2697(02)00695-4

Cited by 54 publications

(36 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Predeveloped human ␤-actin primer sets were purchased from Invitrogen (Carlsbad, CA). Each fluorogenic LUX primer was labeled with a 6-carboxy-fluorescein at the 3Ј end and had a short tail of 4 -6 nucleotides on the 5Ј end complementary to the 3Ј primer end (16,26,27). Primer specificity of amplification was confirmed by gel electrophoresis.…”

Section: Methodsmentioning

confidence: 99%

Induced microsomal PGE synthase-1 is involved in cyclooxygenase-2-dependent PGE2 production in gastric fibroblasts

Shinji

Tsukui

Tatsuguchi

et al. 2005

American Journal of Physiology-Gastrointestinal and Liver Physi

View full text Add to dashboard Cite

.-We have previously shown that the cyclooxygenase (COX)-2/PGE2 pathway plays a key role in VEGF production in gastric fibroblasts. Recent studies have identified three PGE synthase (PGES) isozymes: cytosolic PGES (cPGES) and microsomal PGES (mPGES)-1 and -2, but little is known regarding the expression and roles of these enzymes in gastric fibroblasts. Thus we examined IL-1␤-stimulated mPGES-1 and cPGES mRNA and protein expression in gastric fibroblasts by quantitative PCR and Western blot analysis, respectively, and studied both their relationship to COX-1 and -2 and their roles in PGE2 and VEGF production in vitro. IL-1␤ stimulated increases in both COX-2 and mPGES-1 mRNA and protein expression levels. However, COX-2 mRNA and protein expression were more rapidly induced than mPGES-1 mRNA and protein expression. Furthermore, MK-886, a nonselective mPGES-1 inhibitor, failed to inhibit IL-1␤-induced PGE2 release at the 8-h time point, while totally inhibiting PGE2 at the later stage. However, MK-886 did inhibit IL-1␤-stimulated PGES activity in vitro by 86.8%. N-(2-cyclohexyloxy-4-nitrophenyl)-methanesulfonamide (NS-398), a selective COX-2 inhibitor, totally inhibited PGE2 production at both the 8-h and 24-h time points, suggesting that COX-2-dependent PGE2 generation does not depend on mPGES-1 activity at the early stage. In contrast, NS-398 did not inhibit VEGF production at 8 h, and only partially at 24 h, whereas MK-886 totally inhibited VEGF production at each time point. These results suggest that IL-1␤-induced mPGES-1 protein expression preferentially coupled with COX-2 protein at late stages of PGE2 production and that IL-1␤-stimulated VEGF production was totally dependent on membrane-associated proteins involved in eicosanoid and glutathione metabolism (MAPEG) superfamily proteins, which includes mPGES-1, but was partially dependent on the COX-2/PGE2 pathway.interleukin-1␤; MK-886; MAPEG superfamily; prostaglandin E synthase activity PROSTAGLANDINS (PGs), potent lipid mediators during pain and inflammation, are derived through the cyclooxygenase (COX)-catalyzed conversion of arachidonic acid to PGH2 (5,9,25,33). PGs also play a physiological role in maintaining the integrity of the gastric mucosa, in which COX-1 is constitutively expressed (12,30,35). In addition, we have shown in previous studies that COX-2 is expressed in fibroblasts beneath the gastric ulcer bed in humans (20,45) and that N-(2-cyclohexyloxy-4-nitrophenyl)-methanesulfonamide (NS-398), a selective COX-2 inhibitor, delays gastric ulcer healing in mice (21), suggesting that COX-2 is also important for gastric mucosal integrity (7,31). Furthermore, in a more recent study, we were able to show in vitro that gastric fibroblasts express COX-2 and produce COX-2/PGE2-dependent VEGF in response to IL-1␤, a proinflammatory cytokine (20).Recently, three different PGE synthase (PGES) isozymes have been identified: cytosolic PGES (cPGES) (44), microsomal PGES (mPGES)-1 (11, 18, 23), and mPGES-2 (22, 43). cPGES, is constitutively and ubiquitously ex...

show abstract

Section: Methodsmentioning

confidence: 99%

Induced microsomal PGE synthase-1 is involved in cyclooxygenase-2-dependent PGE2 production in gastric fibroblasts

Shinji

Tsukui

Tatsuguchi

et al. 2005

American Journal of Physiology-Gastrointestinal and Liver Physi

View full text Add to dashboard Cite

show abstract

“…A representative experiment is shown. The DD-Ct method was used to compare samples (Lowe et al 2003).…”

Section: Quantitative Rt-pcrmentioning

confidence: 99%

Poly(A)-binding protein modulates mRNA susceptibility to cap-dependent miRNA-mediated repression

Walters¹,

Bradrick²,

Gromeier³

2009

RNA

View full text Add to dashboard Cite

MicroRNAs (miRNAs) regulate gene expression post-transcriptionally through binding specific sites within the 39 untranslated regions (UTRs) of their target mRNAs. Numerous investigations have documented repressive effects of miRNAs and identified factors required for their activity. However, the precise mechanisms by which miRNAs modulate gene expression are still obscure. Here, we have examined the effects of multiple miRNAs on diverse target transcripts containing artificial or naturally occurring 39 UTRs in human cell culture. In agreement with previous studies, we report that both the 59 m 7 G cap and 39 poly(A) tail are essential for maximum miRNA repression. These cis-acting elements also conferred miRNA susceptibility to target mRNAs translating under the control of viral-and eukaryotic mRNA-derived 59 UTR structures that enable cap-independent translation. Additionally, we evaluated a role for the poly(A)-binding protein (PABP) in miRNA function utilizing multiple approaches to modulate levels of active PABP in cells. PABP expression and activity inversely correlated with the strength of miRNA silencing, in part due to antagonism of target mRNA deadenylation. Together, these findings further define the cis-and trans-acting factors that modulate miRNA efficacy.

show abstract

“…17 Q-PCR with fluorogenic LUX primers was performed to detect mRNA levels of HIF-1a gene in HUVEC cells according to a previously published method. 18 A LUX primer pair for human HIF-1a was designed for Q-PCR using the LUX Designer Software (Invitrogen) and synthesized by Invitrogen. The LUX primer pair included one unlabeled primer and one labeled with the single fluorophore, FAM.…”

Section: Q-pcr With Lux Primersmentioning

confidence: 99%

Antiapoptotic action of hypoxia-inducible factor-1α in human endothelial cells

Liu

et al. 2004

Laboratory Investigation

View full text Add to dashboard Cite

Hypoxia-inducible factor-1 (HIF-1) is the major transcription factor involved in the adaptive response to hypoxia and consists of HIF-1a and HIF-1b subunits. Indirect evidence suggests that HIF-1a may exert both proapoptotic and antiapoptotic actions in response to hypoxia. In this study, we evaluated the effects of RNA interference (RNAi) targeting HIF-1a messenger RNA (mRNA) on apoptosis in primary cultured human umbilical vascular endothelial cells (HUVECs) exposed to anoxia and reoxygenation (A/R). HUVECs were transfected with specific 21-nt small interfering RNA (siRNA) duplexes targeting HIF-1a mRNA sequences or scrambled RNA duplexes and subjected either to normoxia for 251/2 h or to anoxia for 11/2 h, and subsequently normoxia for 24 h (A/R). Control samples were subjected to A/R but not transfected. HUVECs apoptosis was evaluated by Tdt-mediated dUTP nick end-labeling (TUNEL) assay and by activated caspase-3 immunostaining and immunoblotting. The efficacy of RNAi was assessed by knockdown of HIF-1a mRNA and protein expression via in situ hybridization, real-time quantitative PCR, immunohistochemistry, and Western blotting. When compared with normoxic cultures, A/R significantly upregulated HIF-1a mRNA and protein expression in HUVECs, but did not appreciably alter the percentage of apoptotic cells. In contrast, a significantly greater proportion of HUVECs transfected with specific siRNA duplexes and exposed to A/R demonstrated evidence of apoptosis when compared with nontransfected cells. Transfection with specific siRNA duplexes knocked down HIF-1a mRNA and protein expression in A/R-treated cells by approximately 60%, whereas transfection with scrambled siRNA duplexes had no noticeable effect on HIF-1a expression. These findings strongly suggest that HIF-1a exerts an antiapoptotic role in HUVECs stressed by anoxia.

show abstract

Quantitation of gene expression in neural precursors by reverse-transcription polymerase chain reaction using self-quenched, fluorogenic primers

Cited by 54 publications

References 38 publications

Induced microsomal PGE synthase-1 is involved in cyclooxygenase-2-dependent PGE2 production in gastric fibroblasts

Induced microsomal PGE synthase-1 is involved in cyclooxygenase-2-dependent PGE2 production in gastric fibroblasts

Poly(A)-binding protein modulates mRNA susceptibility to cap-dependent miRNA-mediated repression

Antiapoptotic action of hypoxia-inducible factor-1α in human endothelial cells

Contact Info

Product

Resources

About