Premium
Droplet Digital PCR Reveals Substantial Tissue‐Specific Differences in Estrogen Receptor Expression Profiles
Author(s) -
Hutson Dillion D,
Duong Jennifer L.,
Sato Ryousuke,
Lindsey Sarah H.
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.827.11
Subject(s) - gper , estrogen receptor , aromatase , estrogen , biology , medicine , receptor , endocrinology , messenger rna , nucleic acid , kidney , rna , gene , biochemistry , cancer , breast cancer
Our previous work shows an important role for the novel G‐protein Coupled Estrogen Receptor (GPER) in female cardiovascular health. However, the relative importance of this receptor in the context of physiological estrogen signaling has thus far remained elusive, since direct comparison of transcript number has been unattainable due to the relative nature of existing PCR techniques. Droplet digital PCR (ddPCR) is a new technique that allows absolute quantification of nucleic acid concentration from experimental samples. We utilized this technology to compare absolute copy numbers for the three known estrogen receptors (GPER, ERα, ERβ) as well as aromatase, a key enzyme in the synthesis of estrogen. Quantitative ddPCR analysis of purified RNA gathered from cultured A7r5 embryonic rat aortic smooth muscle cells and rat kidney cortex was performed via the BioRad QX200 system for the genes of interest. Expression levels of GPER in A7r5 cells were 10.0 ± 1.4 copies per ng of RNA, while transcript levels for ERα, ERβ, and aromatase were significantly lower at 0.6 ± 0.1, 1.6 ± 0.2, and 2.9 ± 0.7 copies/ng of RNA, respectively (P<0.0001 vs. GPER). Interestingly, in kidney tissue ERα was the predominant transcript at 743.6 ± 4.4 copies in comparison with 30.8 ± 1.4 copies of GPER (P<0.0001). ERβ was expressed at very low levels in kidney (0.15 ± 0.05 copies) while aromatase was undetectable. This data suggests that GPER is expressed at significantly higher levels than nuclear estrogen receptors in A7r5 cells. In contrast, the profile of estrogen receptors in kidney tissue differs greatly, with a predominance of ERα. In conclusion, estrogen responses in different tissues may be modulated by the amounts of these receptors. Moreover, knowledge of these variable receptor profiles can be utilized to target estrogen's protective effects in a tissue‐specific manner. Support or Funding Information National Institute of General Medical Sciences IDeA Program (COBRE, P30GM103337)