Poly a polymerase reaction buffer

Total RNA was extracted using the RNA Simple Total RNA Kit (TIANGEN, DP419), and RNA in the cytoplasm and nucleus was isolated using the Nuclear/Cytoplasmic Isolation Kit (BioVision, San Francisco, CA, United States) according to the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, #K1622) and Poly(A) Polymerase Reaction Buffer (NEB, M0276s) according to the manufacturer’s instructions. According to the manufacturer’s instructions, real-time qRT–PCR analysis was performed using the Platinum SYBR Green qPCR SuperMix-UDG kit (Life Technologies, Gaithersburg, MD, United States). Fold changes in RNA expression were quantified using the 2^−ΔΔCt method. The primer sequences used in this study are listed in Supplementary Table S1.

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 was extracted from selected cells or tissues using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to instructions provided by the manufacturer. Complementary DNA (cDNA) was synthesized using RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, #K1622, city, state, USA) and poly(A) Polymerase Reaction Buffer (NEB, M0276s, city, state, USA) to synthesize complementary DNA (cDNA). Real-time qRT-PCR analysis was performed using the Platinum SYBR Green qPCR SuperMix-UDG kit (Life Technologies, Gaithersburg, MD, USA) according to the instructions provided by the manufacturer. Nucleus/Mass Separation Kit (BioVision, San Francisco, CA, USA) was used to separate RNA from the cytoplasm and nucleus according to the protocols provided by the manufacturer. The 2-ΔΔCt method was used to quantify the ploidy changes in RNA expression. The detailed primer sequences used in this study are listed in Table S2.