Rnase r buffer

The RNase R treatment was performed essentially as described in a previous report [25 (link)]. The purified RNase R enzyme and RNase R buffer were obtained from Epicentre (Epicentre, San Diego, CA, USA). The reaction mixtures for the RNase R treatment contained 4 µg of human skeletal muscle total RNA with or without 40 units of RNase R in 40 µL solutions. The incubation for RNA digestion was performed for 30 min at 37 °C. The samples were subjected to phenol/chloroform extraction, followed by ethanol precipitation. After dissolving the precipitates in water, the nondigested human skeletal muscle total RNA (4 µg) and the RNase R-digested RNA from the same source (4 µg) were used for cDNA synthesis as described above.

Adult brain samples (sample 1–5, Supplementary Materials) were obtained from the Harvard Brain Tissue Resource. Total RNA was extracted from 5 adult frontal lobe samples. To obtain total RNA, tissue pieces were first fixed in OCT and two sections were used for total RNA purification. Total RNA purification for these samples and 6 human blood samples was performed using RiboPureTM RNA purification kit (Ambion) according to the manufacturer instructions. Total RNA from fetal and adult liver, fetal and adult heart, and placenta were purchased from Clonetech. Total RNA from the FFPE tissues was extracted using High Pure FFPET RNA isolation kit (Roche) according to the manufacturer instructions. RNA from the serum was purified using Plasma/Serum Circulating and Exosomal RNA purification Kit (Norgen). RNase R treatment of total RNA was performed after ribosomal RNA depletion. Briefly, 2 μg RNA was added to a mixture of 1x RNase R buffer (Epicentre®), 1 mM MgCl_2, and 1.0 μl (20 U/μl) RNase R enzyme (Epicentre®). The mix was then incubated at 37 C° for 10 minutes. RNase R was then inactivated by adding 1:1 volume of H₂O. All samples are listed in Supplementary Materials.