Mir x mir first strand synthesis kit

The total RNA was isolated from tissues and cell lines using TRIzol reagent (Invitrogen, CA, USA), according to the manufacturer’s protocol. For circRNAs, RNase R was used to degrade linear RNA, which has poly(A), and amplified by divergent primer. cDNA was synthesized from 1 μg of total RNA in 21 μL reaction volumes using oligo(dT) 18 primers and SuperScript reverse transcriptase. PCR amplification was carried out with Taq DNA Polymerase (TaKaRa, Tokyo, Japan) using 1 μL of the first-strand cDNA as template. The amplification reactions were run with 30 thermocycles of 30 s at 94°C, 30 s at 55°C, and 30 s at 72°C. β-Actin was used as an endogenous control. For miR-1179 analysis, miRNA was treated with DNase I to eliminate genomic DNA, and cDNA was synthesized by the Mir-X miR First-Strand Synthesis Kit (TaKaRa). SYBR Premix Ex TaqII (TaKaRa) was used for qRT-PCR. The expression was normalized to RNU6-2. The expression levels were calculated by the 2^˗ΔΔCT method.

The total RNA was isolated from tissues and cell lines using TRIzol reagent (Invitrogen, CA, USA), and exosomal RNA was extracted from plasma and culture medium using the exoRNeasy midi kit (QIAGEN, Valencia, CA, USA) according to the manufacturer’s protocol. For circRNAs, RNase R was used to degrade linear RNA, which have poly(A), and was amplified by divergent primer. Specific divergent primers spanning the back-splice junction site of circRNAs were designed. To quantify the amount of mRNA and circRNA, cDNA was synthesized using a PrimeScript RT reagent kit (TaKaRa, Dalian, China). The qRT-PCR analysis on circRNA and mRNA was performed using a PrimeScript RT reagent kit (TaKaRa) and SYBR premix Ex TaqII (TaKaRa). β-actin was used as an endogenous control. For miR-1178 analysis, miRNA was treated with DNase I to eliminate genomic DNA, and cDNA was synthesized by Mir-X miR first-strand synthesis kit (TaKaRa). SYBR premix Ex TaqII (TaKaRa) was used for qRT-PCR. The expression was normalized to RNU6-2. The 2^−ΔΔCT method was adopted to calculate relative expression.

The total RNA was isolated from tissues and cell lines using TRIzol reagent (Invitrogen, CA, USA), and exosomal RNA was extracted from plasma and culture medium using the exoRNeasy Midi Kit (QIAGEN, Valencia, CA, USA), according to the manufacturer’s protocol. For circRNAs, RNase R was used to degrade linear RNAs, which have poly (A), and amplified by a divergent primer. Specific divergent primers spanning the back-splice junction site of circRNAs were designed. To quantify the amount of mRNA and circRNA, cDNA was synthesized using PrimeScript RT Reagent Kit (TaKaRa, Dalian, China). The qRT-PCR analysis on circular RNA and mRNA was performed using Prime Script RT Reagent Kit (TaKaRa) and SYBR Premix Ex Taq II (TaKaRa). β-Actin was used as an endogenous control. For miR-1178 analysis, miRNA was treated with DNase I to eliminate genomic DNA, and cDNA was synthesized by Mir-X miR First-Strand Synthesis Kit (TaKaRa). SYBR Premix Ex Taq II (TaKaRa) was used for qRT-PCR. The expression was normalized to RNU6-2. The 2^−ΔΔCT method was adopted to calculate relative expression.

All the RNAs were isolated from clinical specimens and cells using TRIzol reagents (Invitrogen, USA). The isolation was carried out according to the kit specifications. For RNA digestion using RNase R (Epicenter Technologies, USA), 4 mg of total RNA was incubated with 6 U/mg RNase R for 20 min. For cellular fractionation, NE-PER nuclear and cytoplasmic extraction reagents (Thermo Scientific, USA) were used to separate the nuclear and cytoplasmic fractions. To detect circRNA and mRNA, two kits were used, that is, SYBR Premix Ex Taq II (TaKaRa, China) and RT reagent kit (TaKaRa, China). Subsequently, the reactions were measured with the Roche LightCycler 480 PCR instrument (Roche, Basel, Switzerland) according to the manufacturer’s specifications. For miRNA qRT-PCR, DNase I was utilized to treat the samples and eliminate gDNA in the samples. The Mir-X miR first-strand synthesis kit (TaKaRa, China) was later used to synthesize cDNA. Lastly, SYBR Premix Ex Taq II (TaKaRa, China) was utilized to perform fluorescence PCR. The primers are listed in Table S2.