M mlv reverse transcription system

Total RNA was extracted from the aortic tissues and purified using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). One microgram of total RNA per sample was then used in a quantitative polymerase chain reaction (qPCR) analysis. Reverse transcription was performed using the M-MLV Reverse Transcription system (Takara Co. Ltd., Dalian, China) under the following conditions: 42°C for 2 min, 95°C for 5 s, and 37°C for 15 min. The resulting cDNA was subjected to qPCR using the SYBR Green reagent (Takara Co. Ltd.) and an ABI 7500 quantitative PCR instrument (Applied Biosystems, Inc., Foster City, CA, USA) under the following conditions: pre-denaturation at 95°C for 10 min, followed by 35 cycles of denaturation at 95°C for 15 s, annealing at 60°C for 25 s, and extension at 72°C for 30 s. Three independent reactions were run per sample. The relative mRNA concentrations were calculated after normalization to the concentration of GAPDH mRNA expression (internal control). The following primers were used: mmu-miR-21-Fwd: 5′-GTCAGGC TAGCTTATCAGA-3′; U6-Fwd: 5′-CTCGCTTCGGCAGCACA-3′, U6-Reverse: 5′-GTATCCAGTGCAGGGTCCGAGGT-3′; PDCD4-Fwd: 5′-AGGTCGTCTTAAACCAGAGAG-3′, PDCD4-Reverse: 5′-ATGTCAGAAATGCCTTGTACC-3′, GAPDH-Fwd: 5′- TCAAGAAGGTGGTGAAGCA-3′, GAPDH-Reverse: 5′-GTCAAAGGTGGAGGAGTGG-3′.

Total RNA was extracted from leaves and roots using the TaKaRa RNAiso reagent, and then treated with RNase-free DNaseI (TaKaRa), following the kit instructions. RNA concentration was measured with a spectrophotometer (NanoDrop 2000, Thermo, USA) at 230, 260 and 280 nm, and the 260/280 nm ratio within the range of 1.80–2.20 and 260/230 nm ratio approximately 2.00 were retained. The first-strand cDNA was synthesized based on 0.5 μg total RNA using M-MLV reverse transcription system (TaKaRa), according to the manufacturer’s instructions. The cDNAs were diluted 1:10 with nuclease-free water prior to the qRT-PCR analyses.

Total RNA from cultured cell were extracted with RNAiso plus (Takara, 9108) following the manufacture’s protocol. For miRNA expression assay, total RNA was reversely transcribed using Taqman MicroRNA Reverse Transcription Kit (Life Technologies, 4366597), then miRNA real-time PCR was performed using Taqman MicroRNA Assay Kit (Life Technologies). For mRNA expression assay, total RNA was reversely transcribed using M-MLV Reverse Transcription System (Takara) and SYBE Green PCR master mix (Toyobo, QPK-201) was purchased for mRNA real-time PCR. All quantitative real-time PCR were performed on an ABI 7300 Real Time System, RNU6 or GAPDH was used as internal control for miRNA and mRNA assay respectively. Relative gene expression was calculated by mean of relative quantification (2^-ΔΔCt) as previously described [60 (link)]. The specific real-time PCR primers for each gene were listed in Table S2 (Additional file 5: Table S2).

Total RNA was isolated according to the RNAiso kit (TaKaRa, Dalian, China) and was then treated with RNase-free DNaseI (TaKaRa). RNA concentration was detected spectrophotometrically (NanoDrop 2000, Thermo, Waltham, MA, USA) at wavelengths of 230, 260, and 280 nm, and the 260/280 nm ratio within the range of 1.80–2.20 and 260/230 nm ratio at approximately 2.00 were obtained. First-strand cDNA was synthesized based on 0.5 μg total RNA using the M-MLV reverse transcription system (TaKaRa), according to the manufacturer’s instructions. The cDNAs were diluted in a 1:20 ratio of CDNA to nuclease-free water prior to the qRT-PCR analyses.