Rnase t1

TNF mRNA ARE binding activity was evaluated with the RNA gel mobility shift assay (GMSA) according to a previous procedure [7] (link) using a TNF mRNA ARE probe (100000−200000 cpm/reaction) with the sequence shown in Figure 7B. The RNA probe was transcribed from mouse TNF mRNA ARE region (nucleotides 1281−1350 of GenBank accession no. X02611) using [α-³²P]UTP (NEN Life Sciences, Boston, MA) and T₇ RNA polymerase with the Promega’s RiboProbe In Vitro Transcription System (Promega Corp., Madison, WI). The binding reaction mixtures were incubated for 30 min at room temperature before digestion with 100 units of RNase T₁ (Epicentre, Madison, WI) for 15 min at 30°C. The TTP−probe complexes and free probes were separated by 6% native PAGE and detected by autoradiography on X-ray film.

RNA fragments digested by RNases were analyzed by mass spectrometry as described previously (31 (link),32 (link)). In brief, 2–5 ng of isolated tRNA was digested with RNase T₁ (Epicentre) or RNase A (Ambion) and analyzed by an LTQ Orbitrap mass spectrometer (Thermo Scientific) with a nano-electrosprayer connected with a splitless nanoflow high pressure liquid chromatography system (DiNa, KYA Technologies). Alternatively, 0.4–2 μg isolated tRNA was digested with RNase T₁ and analyzed on a LCQ DUO ion-trap mass spectrometer with an ESI (electrospray ionization) source (Thermo Finnigan) and HP1100 liquid chromatography system (Agilent Technologies) in negative ion detection mode. Nucleoside analysis was performed as described previously (31 (link)). In brief, about 4 μg of isolated tRNA was digested to nucleosides with nuclease P1 (Wako Pure Chemical Industries) and bacterial alkaline phosphatase C75 (Takara bio), and then analyzed on the LCQ DUO ion-trap mass spectrometer with an ESI source and HP1100 liquid chromatography system in positive-ion detection mode. ProMass (Novatia) was used to obtain the uncharged masses of whole tRNAs by deconvolution of the multiply charged mass spectra.

rRNA (100 fmol of 23S rRNA and 100 fmol of 16S rRNA) was digested at 37°C for 30 min in a 10 μl reaction mixture containing 10mM ammonium acetate (pH 5.3) and 5 U/μl RNase T1 (Epicentre). Subsequently, an equal volume of 0.1M triethylamine-acetate (TEAA) (pH 7.0) was added to the reaction mixture for LC/MS. Analysis of RNA fragments by capillary liquid chromatography (LC) coupled with nano electrospray (ESI) LC/MS was carried out using a linear ion trap-orbitrap hybrid mass spectrometer (LTQ Orbitrap XL, Thermo Fisher Scientific). The nomenclatures for product ions of nucleic acids are those suggested in reference (19 (link)).

To test the protection of RNase by membrane, EVs isolated from 20 ml of HEK293T cell conditioned medium cultured in 10% exosome-depleted FBS or serum-free medium were re-suspended in 150 μl PBS and were equally split into three tubes. To one tube 1% NP40 was added and the vesicles were treated on ice for 15 mins to disrupt the membrane structure (NP40 treated). Then 1.25 μl of proteinase K (20 mg/ml) was added to each tube and incubated at 37°C for 30 mins to digest proteins. Proteinase K was then inhibited by addition of PMSF to 5 mM (no heat inactivation was included to avoid possible membrane damage by heating). Finally, 2 μl RNase T1 (Epicentre, 1 Ku/μl) and 6 μl RNase A (Sigma, 1.25 mg/ml) were added to the NP40 treated tube and the test tube, while 8 μl PBS was added to the control tube. All tubes were incubated at room temperature for 15 mins and then at 37°C for 30 mins. The vesicles were then used for miRNA extraction and RT-PCR analysis. It is critical that the conditioned medium and the vesicles have never been frozen after collection for this experiment.