(A-C) Measurement of miR-16 in ES cells, NIH/3T3 cells, and mouse embryonic fibroblasts (MEFs). (1) Three freeze-thaw cycles followed by 95°C for 5min; (2) Three freeze-thaw cycles only (3) Treatment at 95°C for 5min; (4) Treatment at 4°C only as a control. (D-F) Measurement of miR-20 level in ES cells, NIH/3T3 cells, and MEFs. Treatments 1, 2, 3 and 4 are as specified in A-C. (G) RNase I treatment of ES cell lysates. (1) ES cells after three freeze-thaw cycles, were treated with RNase I for 5min, and following exposure at 95°C for 5min to release all RISC-bond miRNAs. (2) Three freeze-thaw cycles followed by incubation in the buffer for 5min, and at 95°C for 5min to release all RISC-bond miRNAs (as a control for RNase I treatment). (3) Incubation at 95°C for 5min to release miRNAs from RISC complex, followed by RNase I treatment for 5min, and further incubation of the cell lysate at 95°C for 5min. (4) ES cells were incubated at 95°C for 5min to release miRNAs from RISC complex, followed by treatment with buffer treatment for 5min, and incubation at 95°C for 5min (as a control for RNase I treatment). (H) RNase I treatment of MEF (Mouse Embryonic Fibroblast) lysates. (1) MEFs after three freeze-thaw cycles, were treated with RNase I for 5min, and following exposure at 95°C for 5min to release all RISC-bond miRNAs. (2) Three freeze-thaw cycles followed by incubation in the buffer for 5min, and at 95°C for 5min to release all RISC-bond miRNAs (as a control for RNase I treatment). (3) Incubation at 95°C for 5min to release miRNAs from RISC complex, followed by RNase I treatment for 5min, and further incubation of the cell lysate at 95°C for 5min. (4) MEFs were incubated at 95°C for 5min to release miRNAs from RISC complex, followed by treatment with buffer treatment for 5min, and incubation at 95°C for 5min (as a control for RNase I treatment).