Rnaase inhibitor

hPTC transduced with pRetroX-G1-Red (mCherry-G1) retrovirus were trypsinized (Euroclone) at passage 2 after transduction. The cells were then fixed with PFA 0.25%, 0.5% saponin (Merck) with the addition of 1:25 RNAase inhibitor (Promega, N2615). Then anti-DsRed (1:25, Clontech, 632496) or isotype control was incubated for 1 h at RT followed by 1 h incubation with secondary antibody Alexa Fluor 647 goat anti-rabbit (1:100, Thermo Fisher Scientific, A-21245) to detect mCherry⁺ hPTC. All the antibodies were diluted in 0,5% saponin (Merck) with the addition of 1:100 RNAase inhibitor (Applied Biosystems, N8080119). All the solutions were diluted in RNAase-free PBS prepared with DEPC water (Merck). The procedure was carried out on ice. Finally, hPTC were incubated with DAPI (1:1000, Thermo Fisher Scientific) to perform the DNA content analysis and sorted on the FACSAria III BD (Bioscience). Alexa Fluor 647 secondary antibody was excited by a 633 nm laser line, DAPI was excited by a 405 nm laser line. Data were analysed by FacsDiva software (Beckman Coulter).

According to methods reported previously [39 (link)], poly(A)-tailed RNAs were used in real-time reverse transcriptase polymerase chain reaction. In brief, total RNAs isolated from 100 μL of plasma, 20 μM of rATP, 1.5 μL of poly(A) polymerase reaction buffer, and 1 unit poly(A) polymerase (New England Biolabs, Hitchin, UK) were mixed in a 15-μL reaction system and incubated at 37°C for 60 min according to the manufacturer's protocol. Subsequently, poly(A)-tailed small RNA (15-μL total volume) was incubated with 2 μL of reverse-transcriptase primer (5′-GCG AGC ACA GAA TTA ATA CGA CTC ACT ATA GG (T)18(A,G or C)(A,G,C or T) -3′) at 70°C for 5 min to remove any RNA secondary structure. The reactions were chilled on ice for at least 5 min and the remaining reagents, then 8 μL of 5 × ImProm-II reverse transcription buffer, 105 μmol of MgCl2, 20 μmol of dNTP, 40 units of RNAase inhibitor, and 30 units of ImProm-II reverse transcriptase (Promega, Madison, WI, USA), were added according to the protocol. The reaction system was 40 μL, proceeding at 42°C for 30 min, 72°C for 15 min, and 20°C for 5 min.

Total RNA from the cultured cells was extracted using TRIzol reagent (Invitrogen, USA). The nuclear and cytoplasmic fractions were separated using 0.5% NP-40 (Solarbio, Beijing, China) with an RNAase inhibitor (Promega, USA), followed by RNA extraction using TRIzol reagent (Sigma, USA). One microgram of the total RNA was used as template for cDNA synthesis using a PrimeScript RT Reagent Kit (Takara, Japan). Real-time quantitative PCR was performed in triplicate using the SYBR Green reaction mix (Takara, Japan) on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad, USA). The primer sequences used for RT-qPCR are listed in Table S1.