Indexed on: 04 May '05Published on: 04 May '05Published in: Analytical Biochemistry: Methods in the Biological Sciences
Because reproductive tracts in ovariectomized rodents, which are commonly used in endocrinological studies, exhibit drastic changes in response to exogenous estrogens, quantitative evaluation of gene expression requires extra caution. We found that the whole mRNA content of total RNA in the uterus of the ovariectomized rat was dose-dependently reduced by treatment with 17alpha-ethynylestradiol (EE) for 3 consecutive days. Because of the decline in the ratio of mRNA/total RNA, real-time reverse transcriptase-polymerase chain reaction analysis seemingly showed that the relative ratios of the levels of stable RNAs, rRNA18S, rRNA5S, and tRNA-Gly, to whole mRNA were increased by EE. These results indicate that applying a fixed concentration of total RNA and stable RNA as an internal control to uterine mRNA quantification should be reconsidered. On the other hand, the beta-actin gene showed the most stable expression among the housekeeping genes examined. Using beta-actin mRNA as an internal control, we observed that tissue inhibitor of metalloproteinase-3 (TIMP3) mRNA was dose-dependently reduced 24 h after treatment with EE or bisphenol A for 3 days. Continued investigation of TIMP3 with an appropriate internal control may be helpful in elucidating the mechanisms involved in uterine activation.