Hello. I am now making a research proposal about the role of Vit.D (Calcitriol) in pregnancy on cellular level. I understand the theory of calcitriol which will be transfered into nucleus via VDR. VDR then exhibits heterodimerization with RXR. Therefore, Is it correct if I use RT-PCR to measure the expression of calcitriol? Can anyone provide me the protocol? My sample will be placenta tissues.
Thank you
Sincerely.
Interesting question. RT-PCR can be used to analyzed the expression of VDR gene (in your case is placenta tissue) as well as hydroxylation enzymes which involved in calcitriol synthesis (also from extrarenal production).
How much tissue is needed to perform RT-PCR to detect VDR and enzymes you can have a look at recent publications using placenta tissue (1) FASEB J. 2014 May;28(5):2029-37. doi: 10.1096/fj.13-246736. and (2) JCEM 2014 Apr; 99(4): 1348–1356. doi: 10.1210/jc.2013-1366.
Additionally, recent study performed using tissue from human adipose tissue (AT) of women with morbid obesity could also detect Vitamin D3 and 25(OH)D3 content (see JBMR 2017 Feb; 32(2): 237–242). In that paper they mentioned how much tissue they had collected from subjects. Also another study using different technic (see DOI:10.1016/j.jphotobiol.2014.06.008) had detected vitamin D 25(OH)D3 content from human AT, but not calcitriol.
Although enzymes expression (mRNA level) can be detected from several studies using placenta, its not clear about the expression at protein level (functional). Also the short half life of calcitriol has to be taken into account when perform the analysis (to arrange the plan from obtaining of placenta-storage-analysis). Perhaps mass spectro technic (LC-MS) could be used, but how much tissue is needed maybe good to have a look from those papers to compare.
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