qRT-PCR validation of predicted mRNA targets of microRNA. A. qRT-PCR measured mRNA levels of VEGF, ERBB4, SFRP1, and SLC12A1 in 12 ccRCC and matching normal kidney tissue in the validation set. Expression levels, including those of 18S rRNA (control) are plotted on a log-scale relative to a housekeeping gene (beta-actin). These expression level changes agree with predicted changes based on the primary dataset. Dark grey bars denote tumors, while light grey bars denote normal kidney tissue. p-values are for accuracy of discrimination of ccRCC/normal kidney using the t-test. B-E. Agilent chip expression levels of miR-200c, miR-244, miR-34a and miR-21 versus the levels of mRNA that they regulate: VEGFA, ERBB4, SFRP1 and SLC12A1 respectively as measured by qRT-PCR for 12 validation set samples The dark circles represent the values in ccRCC and the light circles in normal kidney. It is clear that loss of mir-200c regulation contributes to an increase in VEGFA transcript while for the other three (tumor suppressor genes), the level of transcript decreases because of a gain in the level of the corresponding microRNA.