Mir x mirna first strand synthesis

RNA extraction and real-time RT-PCR were performed using the same method as previously used^{41 (link)}. Total RNA was extracted from rat liver and primary-cultured hepatocytes using RNAiso Plus (Takara Bio). For miRNA expression analysis, first strand cDNA was synthesized using 1 μg of denatured total RNA at 37 °C for 1 h; reactions were terminated at 85 °C for 5 min with the Mir-X^® miRNA First-Strand Synthesis and SYBR^® qRT-PCR kits (Clontech Laboratories Inc., Mountain View, CA). For mRNA expression analysis, 0.5 μg of denatured total RNA and PrimeScript^® RT reagent kit with gDNA Eraser (Takara Bio) were used to synthesize first strand cDNA; reactions were incubated at 37 °C for 15 min, 84 °C for 5 s, and finally 4 °C for 5 min. Each PCR proceeded via denaturation at 94 °C for 5 s and annealing-extension at 60 °C for 30 s for 40 cycles; SYBR premix Ex Taq (Takara Bio) and specific primers for rat GHR, IGF-1, IGF1R, and GAPDH (Takara Bio, RA009268, RA028844, RA026001, and RA015380, respectively) were used. To normalize each sample for RNA content, GAPDH, a house-keeping gene, or U6 small nuclear RNA (Clontech Laboratories, Inc.) were used for mRNA and miRNA expression analyses, respectively.

The expression profiles of seven selected stress-responsive genes, including OsAGO1 and OsAGO2 salt response genes, were obtained by qRT-PCR. RT primers were designed and listed in Supplement Table 1. Relative expression levels of selected miRNAs were measured by stem-loop RT-PCR as described previously [53 ]. Reverse transcription reactions were then performed with MMLV reverse transcription enzymes (Mir-X miRNA First-Strand Synthesis and SYBR qRT-PCR) to obtain first-strand cDNA. PCR primers (Supplementary Table 1) were designed using Primer3 (http://primer3.ut.ee/) [54 ]. Each reaction was performed in triplicate and OsActin was used as the reference gene. miRNA concentration was set for each reaction with U6 as the internal control. RT-miR168a, the mRQ 3′ primer supplied with the kit, was also used (Mir-X miRNA First-Strand Synthesis and SYBR qRT-PCR, Clontech).

The quantitative real-time PCR was carried out using TaqMan one-step PCR Master Mix (Applied Biosystems, CA, USA). Total RNA was extracted using a TRIzol kit (MDBio Inc., Taipei, Taiwan). The cDNA was reverse transcribed using 2 μg of total RNA with an oligo(dT) primer. Each cDNA (100 ng/25μl reaction) sample was mixed with sequence-specific primers and probes according to the manufacturer's instructions. Sequences for all target gene primers and probes were purchased commercially (Applied Biosystems). β-actin was used as the internal control. The cycling conditions consisted of 10 min of polymerase activation at 95 °C, followed by 40 cycles at 95 °C for 15 s and 60 °C for 60 s.
For miRNA detection, total RNA was extracted using a TRIzol kit (MDBio Inc., Taipei, Taiwan) and expression of miRNA was analyzed using Mir-X miRNA First-Strand Synthesis and Mir-X miRNA qRT-PCR SYBR kits (Clontech Laboratories, Inc., CA, USA). U6 snRNA was used as the internal control. The specific forward primer of miR-381 was 5′-TATACAAGGGCAAGCTCTCTGT-3′. The U6 forward and reverse primers were 5′-CTCGCTTCGGCAGCACATATACTA-3′ and 5′-ACGAATTTGCGTGTCATCCTTGCG-3′. Results were expressed as Ct values and normalized to calculate the average Ct of each sample (ΔCt), and the relative expression of miR-381 was calculated using the comparative Ct method.

The samples were lysed with TRIzol Reagent (Takara, Japan), and total RNA was extracted and quantified to confirm the concentration. In total, 500 ng of RNA was reversely transcribed into cDNA using Prime Script™ RT reagent kit (Takara, Japan). The cDNA was amplified with Ultra SYBR Mixture (CWBIO, Beijing, China) and specific primers by Bio-Rad IQ5 Real-Time System (Bio-Rad, Hercules, CA, USA). Reaction mixtures were treated with pre-denaturation at 95 °C for 10 min, amplified for 40 cycles of denaturation at 95 °C for 15 s, and annealed at 60 °C for 1 min followed by melting curve stage. The relative expression was calculated using 2^−ΔΔCT method. Each reaction was performed in triplicate to determine the expression of target genes, which were normalized against GAPDH. For miRNA, 800 ng of RNA was used to transcribe for cDNA with a reverse transcription kit supplied by Clontech (Mir-X™ miRNA First-Strand Synthesis). RT-PCR was performed with miScript SYBR green PCR kit and miRNA-specific primers, and U6 was recognized as an internal control. All PCR experiments were performed in triplicate. The primer pairs used in the study were listed in Table 1 of supplementary materials.

To validate the expression of the ceRNA regulatory network, the DE-lncRNAs, and their target mRNAs, RT–qPCR validation was performed using SYBR Green assay reagents and a LightCycler^R 480 RealTime PCR System (Roche, USA) as described by Zhou et al. (2012) (link). Total and small RNAs were extracted using the same samples used for RNA-sequencing. For small RNA reverse transcription, universal primers (Supplementary Table S1) in the Mir-X miRNA First-Strand Synthesis (Clontech Laboratories, CA, USA) were used for reverse transcription. Mature miRNA sequences and universal primers (Supplementary Table S1) were used for RT–qPCR according to the manufacturer’s instructions for the SYBR qRT-PCR kit (Clontech Laboratories, CA, USA). For RNA reverse transcription, 2 μg of total RNA was reverse-transcribed using the PrimeScript™ RT Reagent Kit (TaKaRa BIO, Japan). The specific primers of the tested genes are shown in Supplementary Table S1. Each DE-lncRNA, DE-miRNA, and DE-mRNA was analyzed in three replicates. Relative expression levels were calculated using the 2^–ΔΔCt method. Actin was used as the endogenous reference genes for DE-lncRNAs and DE-mRNAs, and 5.8S rRNA was used as the endogenous reference gene for DE-miRNAs (Supplementary Table S1).