Rnase a

Bovine RNase A was purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA, Cat. #: EN0531). RNase A was added in a final concentration of 1 µg/mL to the sample directly before starting incubation at 37 °C and 500 rpm in a ThermoMixer^® Comfort 2000 (Eppendorf, Hamburg, Germany). Digests were stopped by placing samples on ice and adding the RNase inhibitor-including qScript™ XLT One Step RT-qPCR ToughMix^®. Immediately afterwards, the preparations for RT-qPCR analysis were performed with these samples as described in Section 2.3.
Calculation of intact siRNA percentage after incubation was done as follows: ΔC_t values of incubated samples were calculated by subtracting from their C_t values the C_t of non-incubated control reactions which had the same initial target siRNA concentration and were run in the same experiment. The RT-qPCR efficiency (E) was calculated by the Rotor-Gene Q Series software, measuring C_t shifts between different concentrations of the dilution series which was run in the same experiment. A theoretical efficiency of 2 would indicate a 100% increase in target sequences per cycle during RT-qPCR. The percentage of intact siRNA (I_%) was then calculated using the formula: $${\mathrm{I}}_{\%}=\frac{100}{{\mathrm{E}}_{ }^{{\Delta \mathrm{C}}_{\mathrm{t}}}}$$
I_%—Percentage of intact siRNA after incubation
E—RT-qPCR efficiency
ΔC_t—Difference between C_t values of incubated samples and non-incubated controls

Clarified cell lysates were treated with RNase A (Ambion) using the following condition – 1 µg RNase A was added per 100 µg RNA in a 250 µl reaction volume, shaken at 500 rpm (20 min, 25 °C) on a table-top thermo-mixer (Eppendorf); the reaction was quenched by the addition of SUPERase•In RNase inhibitor (~200 units per 100 µg RNA). RNase A digested lysates were layered on top of 10–35% sucrose gradients and processed as described above.