Fig. 3.

Tumor-derived miRNAs are detectable in plasma. (A) Schema for 22Rv1 human prostate cancer xenograft experiment. (B) MiRNAs are present in plasma of healthy control mice and their levels are not nonspecifically altered in cancer-bearing mice. Plasma levels of miR-15b, miR-16, and miR-24 were measured in 12 healthy control mice and 12 xenograft-bearing mice. The mature sequence of these miRNAs is perfectly conserved between mice and humans. Ct values were converted to absolute number of copies/μl plasma by using a dilution series of known input quantities of synthetic target miRNA run on the same plate as the experimental samples (dilution curves are provided in Fig. S2). Values shown have been normalized by using measurements of C. elegans synthetic miRNA controls spiked into plasma after denaturation for RNA isolation (details of the normalization method are provided in SI Text). (C) Tumor-derived miRNAs are detected in plasma of xenograft-bearing mice and can distinguish cancer-bearing mice from controls. Plasma levels of miR-629* and miR-660 (two human miRNAs that are expressed in 22Rv1 cells and do not have known murine homologs) were measured in all control and xenografted mice. Ct values were converted to absolute number of copies/μl plasma and normalized as described for B (see Table 5) threshold. Given that homologous miRNAs are not believed to exist in mice, the low level of signal detected for a few mice in the control group, particularly for the miR-660 assay, is likely to represent nonspecific background amplification. As expected, in the control (nontumor-bearing) mice group, qRT-PCR for miR-629* or miR-660 in plasma from most animals could not detect any appreciable signal. These points are therefore not shown on the graph, even though plasma samples from the entire group of 12 mice in the control group were studied.