Extraction of nucleic acids (RNA and DNA)

RNA and DNA were extracted from each sample. All samples had appropriate amounts of chloroform added to the TRIzol Reagent/TRIzol LS Reagent as per manufacturer’s instructions. The aqueous layer that formed after centrifugation at 12,000×g for 15 min at 4 °C was collected and combined with 1.5× volume 100% ethanol. Purification of microRNA and total RNA from this fraction was performed using miRNeasy Mini/Micro Kit (Qiagen #217004/#217,084) as per manufacturer’s instructions. On-column DNase digestion was performed using the RNase-Free DNase Set (Qiagen #79254).

After the aqueous phase was collected for extraction of RNA, the remaining interphase and phenol chloroform phase was used to extract DNA with the addition of Back Extraction Buffer (BEB - 4 M guanidine thiocyanate, 50 mM sodium citrate, 1 M Tris), 0.25 ml BEB per 0.5 ml TRIzol. The solution was shaken vigorously for 15 s, incubated at room temperature for 10 min and then centrifugated at 12,000×g for 15 min at 4 °C. The resulting aqueous layer was collected, mixed with 1× volume of 100% ethanol, and the DNA was purified using DNeasy Blood & Tissue Kit (Qiagen #69506) as per manufacturer’s instructions.

RNA/DNA was quantified using NanoDrop Lite (Thermo Fisher).