Evaluation of housekeeping genes in placental comparative expression studies

Meller, Margaret; Vadachkoria, Surab; Luthy, David A.; Williams, Michelle A.

doi:10.1016/j.placenta.2004.09.009

Cited by 199 publications

(134 citation statements)

References 19 publications

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“…The amount of SNAT2 RNA was calculated using the 2 -∆Ct method relative to the hypoxanthine-guanine phosphoribosyltransferase (HPRT) and succinate dehydrogenase complex-subunit A (SDHA) housekeeping genes, which were selected from a pool of tested housekeeping genes because they showed a similar amplification efficiency in a scale of RNA concentrations (38). All the assays were provided by Applied Biosystems.…”

Section: Snat2 Gene Expression: Quantitative Real-time Pcr Analysis mentioning

confidence: 99%

SNAT2 expression and regulation in human growth-restricted placentas

et al. 2013

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Background: Amino acid placental delivery is reduced in human intrauterine growth-restricted (IUGR) fetuses, and the activity of placental amino transporters has been consistently shown to be decreased in in vitro studies. We hypothesized lower placental expression and localization of sodium-coupled neutral amino acid transporter 2 (SNAT2 (also known as SLC38A2)), altered levels of intron-1 methylation, and altered distribution of single-nucleotide polymorphisms in human IUGR vs. normal pregnancies. Methods: We studied 88 IUGR and 84 control placentas from singleton pregnancies at elective caesarean section. SNAT2 expression was investigated by real-time PCR and immunohistochemistry. Intron-1 methylation levels were analyzed by pyrosequencing, and single-nucleotide polymorphism distribution was analyzed by allelic discrimination. results: mRNA levels were significantly decreased in IUGR placentas with reduced umbilical blood flows. Syncytiotrophoblast immunostaining was lower in IUGR placentas than in control placentas. Methylation levels were steadily low in both IUGR and control placentas. SNP genotype and allele frequencies did not differ between the two groups. conclusion: This is the first study investigating SNAT2 expression and regulation mechanisms in human IUGR placentas. We confirm previous results obtained in rats and cell cultures that support the fundamental role of SNAT2 in fetal growth and well-being, as well as a possible role of oxygen levels in regulating SNAT2 expression, indicating the relevance of hypoxia in IUGR.

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Section: Snat2 Gene Expression: Quantitative Real-time Pcr Analysis mentioning

confidence: 99%

SNAT2 expression and regulation in human growth-restricted placentas

et al. 2013

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“…Values obtained for each gene were normalized to the geometric mean of 3 housekeeping genes: TBP, SDHA, YWHAZ and/or ACTB (21). A comparative Ct method (∆∆CT method) (22) was applied to the raw Ct values to find a relative gene expression.…”

Section: Nm_003194mentioning

confidence: 99%

Genes responsible for vaginal extracellular matrix metabolism are modulated by women's reproductive cycle and menopause

Shynlova

Bortolini²,

Alarab

2013

Int. braz j urol.

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Objectives: To analyze the expression of genes involved in extracellular matrix (ECM) biogenesis and remodeling in vaginal tissue of women with clinically normal pelvic floor support (defined as controls) according to the phase of menstrual cycle and postmenopausal women with and without pelvic organ prolapse (POP). Materials and Methods:This study examined the expression of matrix metalloproteinases (MMPs), their tissue inhibitors (TIMPs), and the Lysyl oxidase (LOX) family genes in the anterior vaginal wall of Caucasian women by real-time RT-PCR. Initially, mRNA expression was assessed in premenopausal controls in the secretory (group 1, n = 10) vs. proliferative (group 2, n = 8) phase of menstrual cycle. In addition, we compared premenopausal controls in the proliferative phase (group 2) vs. postmenopausal controls (group 3, n = 5). Finally, we analyzed postmenopausal controls (group 3) vs. postmenopausal women with advanced POP (group 4, n = 13). Results: According to the phase of menstrual cycle, MMP1 was significantly reduced (p = 0.003), whereas the expression of TIMP1 and LOXL4 was significantly up-regulated during proliferative phase (both p < 0.01) when compared to the secretory phase in premenopausal control women. Regarding menopausal status/ageing, all MMPs were down--regulated, while TIMP3, TIMP4 and LOXL2 were significantly up-regulated in postmenopausal control women when compared to premenopausal controls (p = 0.005, p = 0.01 and p < 0.001, correspondingly). TIMP4 and LOXL2 mRNA levels were significantly decreased in postmenopausal POP patients compared to asymptomatic postmenopausal controls (p < 0.01 for both). Conclusions: Our results indicate that ovarian cycle and age-related changes influence the expression of genes encoding proteins responsible for ECM metabolism in human vagina. Moreover, POP is associated with alteration in vaginal ECM components after menopause.

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“…For TATA box binding protein (TBP) analysis, cDNA was diluted 1:20, and for TLR analysis cDNA was used undiluted. As previously suggested (Meller et al, 2005), TBP was found to be a suitable reference gene for this study by comparison of twelve common reference genes (data not shown), and purchased from Sigma-Aldrich. These primers have been used in our laboratory for TLR gene expression studies in other cell types ( (Grimstad et al, 2011) and unpublished observations).…”

Section: Quantitative Rt-pcr (Rt-qpcr)mentioning

confidence: 99%