Development of a high sensitivity, nested Q-PCR assay for mouse and human aromatase

Liu, Giujian; Wu, Yu Sheen; Brenin, David R.; Yue, Wei; Aiyar, Sarah E.; Gompel, Anne; Wang, Ji Ping; Tekmal, Rajeshwar Rao; Santen, Richard J.

doi:10.1007/s10549-007-9792-4

Cited by 9 publications

(7 citation statements)

References 33 publications

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“…Various mechanisms such as rate of transcription, half life of message, rate of translation, and half life of protein stability could result in the increased aromatase protein demonstrated by immunohistochemistry. We utilized a nested PCR assay [30] to quantitate aromatase message in paired dense and non-dense tissue. As this is highly labor intensive, assays were performed in a pilot fashion on a small sample of participants (n=13).…”

Section: Discussionmentioning

confidence: 99%

Aromatase immunoreactivity is increased in mammographically dense regions of the breast

Vachon

Sasano

Ghosh

et al. 2010

Breast Cancer Res Treat

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Mammographic breast density is one of the strongest risk factors for breast cancer. Unfortunately, the biologic basis underlying this association is unknown. This study compared aromatase expression or immunoreactivity (IR) in core biopsies from mammographically dense versus non-dense regions of the breast to examine whether estrogen synthesis in the breast is associated with mammographic breast density (MBD) and one possible mechanism through which it may influence breast cancer. Eligible participants were 40+ yrs, had a screening mammogram with visible MBD and no prior cancer or current endocrine therapy. Mammograms were used to identify dense and non-dense regions and ultrasound-guided core biopsies were performed to obtain tissue from these regions. Immunostaining for aromatase employed the streptavidin-biotin amplification method and #677 mouse monoclonal antibody. Aromatase IR was scored in terms of extent and intensity of staining for each cell type (stroma, epithelium, adipocytes) on the histologic section. A modified histological (H)-score provided quantitation of aromatase IR in each cell type and overall. Repeated measures analyses evaluated average differences (βH) in H-score in dense versus non-dense tissue within and across cell types. Forty nine women mean age 50 yrs (range: 40 to 82), participated. Aromatase IR was increased in dense (vs. non-dense) tissue in both the stroma (βH =0.58) and epithelium (βH =0.12) (p<0.01). Adipocytes from non-dense tissue, however, had a greater IR compared to those from dense tissue (βH =-0.24, p<0.01). An overall H-score which integrated results from all cell types demonstrated that aromatase IR was twice as great for dense (mean H-score=0.90, SD=0.53) vs. non-dense (mean H-score=0.45, SD=0.39) breast tissue (βH =0.45; p<0.001). Overall, aromatase IR was greater for mammographically dense vs. non-dense tissue and may partly explain how MBD influences breast cancer.

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Section: Discussionmentioning

confidence: 99%

Aromatase immunoreactivity is increased in mammographically dense regions of the breast

Vachon

Sasano

Ghosh

et al. 2010

Breast Cancer Res Treat

Self Cite

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“…As only a few cells of breast tissue samples can be obtained by FNA [7], a highly sensitive method for detecting mRNA in the breast tissue aspirates is critical. A sensitive nested Q-PCR developed by us [8] and common non-nested Q-PCR were compared using the FNA samples. Our data showed that samples containing trace mRNA were easily detected with nested Q-PCR.…”

Section: Discussionmentioning

confidence: 99%

“…Because of a very small amount of breast tissue obtained by FNA, the challenge is to develop a method that is sensitive and specific enough to detect the mRNA expression levels of aromatase and steroid sulfatase. Previously, we established a nested Q-PCR assay for human aromatase and successfully analyzed several ductal lavage fluid samples [8].…”

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confidence: 99%

Aromatase and steroid sulfatase mRNA expression in fine needle aspirates from benign and malignant breast disorders

Gao

Liu

et al. 2012

Chin. Sci. Bull.

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The fine needle aspiration (FNA) test is a convenient and tolerable technique with minimal invasion that is accepted by most women. Local estrogen synthesis depends mainly on the aromatase and steroid sulfatase pathways that are believed to play important roles in breast carcinogenesis. However, little is known about the level of aromatase and steroid sulfatase mRNA expression in FNA samples which contain only small amounts of tissue. The nested Q-PCR assay has been proven to be a highly sensitive and specific method to assess the aromatase expression of breast tissue. In this study, aromatase and steroid sulfatase mRNA expression in 74 patients with benign or malignant disorders was evaluated and compared using nested Q-PCR and non-nested Q-PCR assays. The expression levels were analyzed and correlated with clinical parameters. No difference in the aromatase expression levels between nested and non-nested Q-PCR was noticed. Age and aromatase mRNA expression level were two independent risk factors for breast cancer (P=0.04 and P=0.00, respectively), while menopausal status and steroid sulfatase mRNA expression levels were not associated with breast cancer. This study showed that both nested and non-nested Q-PCR assays were effective methods for research using FNA breast samples.aromatase, steroid sulfatase, breast disorders, fine needle aspirate, nested Q-PCR

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“…The primers used are listed in Table II . Relative expression levels of total ERβ were calculated by N = 2 – Δ Ct(GAPDH – ERβ) [ 18 ], where N is the relative quantity of mRNA expression.…”

Section: Methodsmentioning

confidence: 99%

Estrogen receptor β promoter methylation: a potential indicator of malignant changes in breast cancer

Gao

et al. 2016

aoms

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IntroductionEstrogen receptor β (ERβ) always lacks expression in estrogen-dependent tumors, which may result from gene inactivation by methylation. In this study, we aimed to determine whether aberrant methylation of the ERβ promoter is associated with decreased ERβ gene expression in breast cancer.Material and methodsERβ methylation status was determined for 132 pairs of breast cancer and adjacent normal tissues via the MethyLight method. Additionally, mRNA relative expression was quantified by real-time polymerase chain reaction (RT-PCR) to determine whether aberrant methylation had a negative correlation with expression. The correlation of ERβ promoter methylation and clinical parameters is also discussed.ResultsMethylation was observed in 96 (72.7%) breast cancer samples, and the median percentage of fully methylated reference (PMR) among methylated tissues was 0.83. Meanwhile, 94 (71.2%) adjacent normal tissues were methylated and the median PMR was 0.48. Compared to adjacent normal tissues, the methylation level of breast cancer was significantly higher (p < 0.001) and mRNA expression was much lower (p < 0.001). There was a significant correlation between ERβ methylation and mRNA expression in adjacent normal breast tissues (p = 0.004). In addition, the methylation rate of cancer tissues whose maximum diameter < 3 cm was significantly higher than those > 3 cm (p = 0.025).ConclusionsERβ promoter methylation level varies between cancerous and adjacent normal breast tissues. There was significant downregulation of ERβ methylation expression in pre-cancerous stages of breast cancer. Therefore, demethylation drugs may offer a potential strategy for preventing the development of pre-cancerous cells.

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Development of a high sensitivity, nested Q-PCR assay for mouse and human aromatase

Cited by 9 publications

References 33 publications

Aromatase immunoreactivity is increased in mammographically dense regions of the breast

Aromatase immunoreactivity is increased in mammographically dense regions of the breast

Aromatase and steroid sulfatase mRNA expression in fine needle aspirates from benign and malignant breast disorders

Estrogen receptor β promoter methylation: a potential indicator of malignant changes in breast cancer

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