Validation of RAKE using real-time PCR. Fluorescence signals from each of the 45 PCR cycles were collected and converted into log10 values. The log10 fluorescence values (Y-axis) of each sample are then plotted against the PCR cycles (X-axis) to generate a sigmoid curve. CT (threshold-cycle; dotted line) determines the minimum PCR cycle required for the reaction to give a threshold fluorescence signal. Samples with more templates require fewer PCR cycles to reach the threshold. Comparison of the miRNA expression levels in pNL4-3- (green curve) and mock-transfected HeLa cells (red curve) are facilitated by using cellular small nuclear U6 RNA (blue curve from mock, and orange curve from pNL4-3; please note that the blue and orange control curves superimpose closely on top of each other, supporting the validity of the PCR conditions for comparing the experimental curves) and an empirically established unchanged miRNA (mi-526c; mock and pNL4-3 samples are shown in red and in green curves, respectively) as normalization references (A). Selected pNL4-3-downregulated miRNAs [miR-93 (B), miR-148b (C), miR-221 (D) and miR-16 (E)] were validated by real-time PCR. Real-time PCR curves for U6 RNA control (mock and pNL4-3) are included in all of the graphs for normalization. Signals of the selected miRNAs measured in the RAKE assay from different samples (1, 2 and 3) are presented in table form at the top of each graph.