Competitive RT-PCR to quantify CFTR mRNA in human endometrium

Mularoni, Angélique; Adessi, G. L.; Arbez-Gindre, Francine; Agnani, G.; Nicollier, M.

doi:10.1093/clinchem/42.11.1765

Cited by 14 publications

(9 citation statements)

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“…In this method, the calibrator and target were amplified with different primers. Furthermore, some of the studies used an exogenous target-like IS RNA or DNA using dilutions of the IS for the quantification of the target mRNA (1,23,27,41). More recently, the quantification of the target mRNA has been carried out using a constant amount of IS in each sample and an external calibration curve consisting of the dilutions of calibrator and a constant amount of IS in each (2,28,38,39,42,44).…”

Section: Discussionmentioning

confidence: 99%

“…Table 2 summarizes some studies (2,23,27,28,38,39,42,44) that report the precision or the detection range of their QRT-PCR assays and that utilize an exogenous IS (panel A) and/or an external calibration curve (panel B). There are many RT-PCR assays each utilizing its own principle, which makes the direct comparison of different assays quite difficult.…”

Section: Discussionmentioning

confidence: 99%

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Dual-Label Detection of Amplified Products in Quantitative RT-PCR Assay Using Lanthanide-Labeled Probes

et al. 2001

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Quantitative RT-PCR (QRT-PCR) enables the sensitive and specific detection of mRNA with a small copy number. We used the QRT-PCR method and dual-label analysis of amplification products for the detection of prostate-specific antigen (PSA) mRNA. The QRT-PCR assay employed a PSA-like internal standard (IS) mRNA, which was used to quantify the PSA mRNA copies and to control the variations during the whole assay procedure from the RNA extraction to the detection of QRT-PCR amplification products by hybridization assay. After co-amplification, the PSA and IS products were detected in a microplate using Eu3+ chelate-labeled PSA and Tb3+ chelate-labeled IS hybridization probes. The detection probes allowed the simultaneous and dual-label detection of PSA and IS products in the same microtiter well. Compared to the single-label assay, the dual-label detection improved the within- and between-assay CV% from 21.7 to 7.5 and from 36.0 to 30.3, respectively. The between- and within-assay variation of the dual-label assay was further studied using PSA-producing LNCaP cells. The cells were found to express 980 +/- 170 (mean +/- SD) copies of PSA-mRNA with the within-assay CV% of 17.7 and 890 +/- 220 (mean +/- SD) copies of PSA-mRNA with the between-assay CV% of 25.0. The methodology developed may help in future studies to obtain reliable quantification of PSA mRNA generated by circulating prostate cancer cells.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Dual-Label Detection of Amplified Products in Quantitative RT-PCR Assay Using Lanthanide-Labeled Probes

et al. 2001

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“…CTFR is expressed in the endometrial epithelium of guinea-pigs and other animals. In human endometrium, CFTR is also widely expressed, and its expression changes in a cyclic manner [43,51]. In cultured glandular cells, it was found to be upregulated by progesterone and downregulated by estradiol [51][52][53].…”

Section: Cystic Fibrosis Transmembrane Conductance Regulator (Cftr) Amentioning

confidence: 99%

“…In human endometrium, CFTR is also widely expressed, and its expression changes in a cyclic manner [43,51]. In cultured glandular cells, it was found to be upregulated by progesterone and downregulated by estradiol [51][52][53]. The expression and role of CFTR in endometriosis has been evaluated by Huang et al [45]: in ectopic, endometrial-like samples, quantitative real-time polymerase chain reaction (qPCR) results demonstrated a significantly higher expression of CFTR mRNA and proteins in endometriotic lesions compared to normal endometria.…”

Section: Cystic Fibrosis Transmembrane Conductance Regulator (Cftr) Amentioning

confidence: 99%

Ion Channels in The Pathogenesis of Endometriosis: A Cutting-Edge Point of View

Riemma

Laganà

Schiattarella

et al. 2020

IJMS

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Background: Ion channels play a crucial role in many physiological processes. Several subtypes are expressed in the endometrium. Endometriosis is strictly correlated to estrogens and it is evident that expression and functionality of different ion channels are estrogen-dependent, fluctuating between the menstrual phases. However, their relationship with endometriosis is still unclear. Objective: To summarize the available literature data about the role of ion channels in the etiopathogenesis of endometriosis. Methods: A search on PubMed and Medline databases was performed from inception to November 2019. Results: Cystic fibrosis transmembrane conductance regulator (CFTR), transient receptor potentials (TRPs), aquaporins (AQPs), and chloride channel (ClC)-3 expression and activity were analyzed. CFTR expression changed during the menstrual phases and was enhanced in endometriosis samples; its overexpression promoted endometrial cell proliferation, migration, and invasion throughout nuclear factor kappa-light-chain-enhancer of activated B cells-urokinase plasminogen activator receptor (NFκB-uPAR) signaling pathway. No connection between TRPs and the pathogenesis of endometriosis was found. AQP5 activity was estrogen-increased and, through phosphatidylinositol-3-kinase and protein kinase B (PI3K/AKT), helped in vivo implantation of ectopic endometrium. In vitro, AQP9 participated in extracellular signal-regulated kinases/p38 mitogen-activated protein kinase (ERK/p38 MAPK) pathway and helped migration and invasion stimulating matrix metalloproteinase (MMP)2 and MMP9. ClC-3 was also overexpressed in ectopic endometrium and upregulated MMP9. Conclusion: Available evidence suggests a pivotal role of CFTR, AQPs, and ClC-3 in endometriosis etiopathogenesis. However, data obtained are not sufficient to establish a direct role of ion channels in the etiology of the disease. Further studies are needed to clarify this relationship.

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“…Mutations of CFTR result in cystic fibrosis (CF), a lethal genetic disease with multi-organ defects, including infertility [ 18 , 19 ]. In the female reproductive tract, CFTR is expressed in the vagina, cervix, uterus, fallopian tube and ovary [ 20 – 22 ]. During the estrus cycle, the expression of CFTR in mouse endometrium is upregulated by estrogen [ 23 ] and downregulated by progesterone [ 24 ].…”

Section: Introductionmentioning

confidence: 99%

Upregulation of CFTR in patients with endometriosis and its involvement in NFκB-uPAR dependent cell migration

et al. 2017

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Endometriotic tissues exhibit high migration ability with the underlying mechanisms remain elusive. Our previous studies have demonstrated that cystic fibrosis transmembrane conductance regulator (CFTR) acts as a tumor suppressor regulating cell migration. In the present study, we explored whether CFTR plays a role in the development of human endometriosis. We found that both mRNA and protein expression levels of CFTR and urokinase-type plasminogen activator receptor (uPAR) were significantly increased in ectopic endometrial tissues from patients with endometriosis compared to normal endometrial tissues from women without endometriosis and positively correlated. In human endometrial Ishikawa (ISK) cells, overexpression of CFTR stimulated cell migration with upregulated NFκB p65 and uPAR. Knockdown of CFTR inhibited cell migration. Furthermore, inhibition of NFκB with its inhibitors (curcumin or Bay) significantly reduced the expression of uPAR and cell migration in the CFTR-overexpressing ISK cells. Collectively, the present results suggest that the CFTR-NFκB-uPAR signaling may contribute to the progression of human endometriosis, and indicate potential targets for diagnosis and treatment.

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Competitive RT-PCR to quantify CFTR mRNA in human endometrium

Cited by 14 publications

References 0 publications

Dual-Label Detection of Amplified Products in Quantitative RT-PCR Assay Using Lanthanide-Labeled Probes

Dual-Label Detection of Amplified Products in Quantitative RT-PCR Assay Using Lanthanide-Labeled Probes

Ion Channels in The Pathogenesis of Endometriosis: A Cutting-Edge Point of View

Upregulation of CFTR in patients with endometriosis and its involvement in NFκB-uPAR dependent cell migration

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