Mirna first strand cdna synthesis

Total RNA from tissues or cells was isolated using an RNA isolation kit (QIAGEN, Qiagen GmbH, Hilden, Germany). mRNA wasreverse‐transcribed to cDNA using a PrimeScript RT reagent Kit (RR047A, Takara Holdings Inc., Kyoto, Japan), whereas miRNA was reverse‐transcribed to cDNA with a miRNA First Strand cDNA Synthesis (Tailing Reaction) kit (B532451‐0020, Sangon Biotech Co., Ltd., Shanghai, China). Next, real‐time qPCR was conducted using a Fast SYBR Green PCR (Applied Biosystems, Foster, CA, USA) on an ABI 7500 real‐time PCR System (Applied Biosystems, Inc., Carlsbad, CA, USA). The primers are listed in Table 1, in which glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) and U6 were used as the endogenous controls. Relative gene expression was evaluated using the 2^–ΔΔCt method.

Total RNA was obtained from MC3T3-E1 using Trizol (Takara). The M-MuLV First Strand cDNA Synthesis Kit and miRNA first Strand cDNA Synthesis (Sangon Biotech) was applied to produce cDNA. The 2 × SG Fast qPCR Master Mix was used to detect relative mRNA or miRNA levels on a system following the manufacturer. Quantified by the 2-ΔΔCt method. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and U6 were applied as controls. All sequences of primers were presented in Table 1.

An miRNeasy Mini Kit (Qiagen) was used to extract total RNA, containing miRNA, from mouse embryos or mouse neural stem cells (C17.2). Extracted RNA was reverse‐transcribed using a miRNA First Strand cDNA Synthesis (Sangon). qRT‐PCR was performed as follows: pre‐denaturation at 95°C for 5 minutes; followed by 45 cycles of 95°C for 15 s and 60°C for 45 s using a SYBR Premix Ex Taq kit (Takara) on a 7500 Real‐time PCR system (ABI). The relative expression of miRNAs and cDNA was quantified using the 2^−ΔΔCt method.

After the cells were rinsed with PBS at 4°C, total mRNA was extracted using Trizol^® reagent and reverse transcribed into cDNA by PrimeScript^TM RT reagent Kit (RR037A). For miRNAs analysis, miRNA were reverse transcribed to cDNAs using the miRNA First-Strand cDNA Synthesis (Sangon, China) with the Stem-loop Method by primers (Supplementary Table 2). RT- qPCR was performed using SYBR^® Primix Ex Taq^TM by primers (Supplementary Table 3). U6 small nuclear RNA and GAPDH was used as an endogenous control for miRNA and mRNA. Relative differences in the PCR product amounts were evaluated by using the 2^–ΔΔCT method.

Total RNA was extracted with TRIzol Reagent (Cat. No: 15596018; Invitrogen, Thermo Fisher Scientific, Wilmington, DE, USA), and its purity was determined using the NanoDrop spectrophotometer (ND-2000; Thermo Fisher Scientific, Waltham, MA, USA). The RNA samples with high purity were reversely transcribed into complementary DNAs (cDNAs) using the TAKARA PrimeScript™ RT reagent kit with gDNA Eraser (Perfect Real Time) (Cat. No: RR047A; Takara, Dalian, China) according to the manufacturer's instruction. In order to determine miRNA-203 expression, the microRNAs were purified using the SanPrep Column microRNA Extraction Kit (Cat. No: B518811; Sangon Biotech (Shanghai) Co., Ltd., China) and the first-strand cDNA synthesis was carried out using miRNA First-Strand cDNA Synthesis (Tailing Reaction) (Cat. No: B532451; Sangon Biotech (Shanghai) Co., Ltd., China) according to the manufacturer's protocol. PCR amplification was carried out in a 20 μL reaction system, which contained 2 μL cDNA, 10 μL SYBR Premix Ex Taq II (TaKaRa, Otsu, Shiga, Japan), and 10 μM of both sense and antisense primers. The expressions were calculated using the 2^−ΔΔCT method. Experiments were performed in triplicate. Primers used here are presented in Table 1.

Total RNA was extracted by Trizol reagent. mRNA and miRNA were reverse transcribed with the PrimeScript™RT reagent Kit with gDNA Eraser and miRNA first strand cDNA synthesis (Sangon Biotech, Shanghai, China) respectively. The primer sequences were designed and synthetized (Table 2). Quantitative real-time PCR (qRT-PCR) was conducted with SYBR Premix Ex Taq™ II. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or U6 was used to respectively normalize the mRNA expression or miRNA expression. The data were compared to normalized control values.