Stepone qrt pcr system

The expression of MiR137-3p, MiR137-5p, MiR34a-5p, MiR203-3p, MiR203-5p, MiR493-3p and MiR493-5p and mRNA of 4 select DEGs were assessed by quantitative reverse transcription PCR (qRT-PCR). Reactions were carried out using a StepOne qRT-PCR system (Applied Biosystems). Fold-change values were calculated using the ∆∆Ct method [91 (link)] relative to the housekeeping genes RNU6 and RNU66 (miRNA) or HPRT (mRNA). A mean expression score for all 7 miRNAs was calculated as the mean ∆∆Ct value across miRNAs for each mouse.

Quantitative real-time PCR was performed as described previously [26 (link)]. The BCP (1-bromo-3-chloropropane) protocol [27 (link)] was applied to extract RNA from transgenic Raphanus sativus L. leaves. The isolated RNA was subjected to DNase-1 digestion before proceeding to cDNA synthesis. First-strand cDNA synthesis was implemented as previously described [28 (link)]. RT-PCR was performed using the StepOne qRT-PCR system (Applied Biosystems, Waltham, MA, USA) following the manufacturer’s instructions in the presence of SYBR Green (SYBR1 GreenERTM qPCR SuperMixes; Karlsruhe, Germany). Primers were purchased from Intron Biotechnology Inc. (Kyungki-Do, Korea). 5′-CTG AAA CCA TCC CTG TCC TC 3′ and 5′-TCT AGG AGG GTC TCA TCC CA-3′ primers were used to detect IFN-α2a transcripts. For the detection of the housekeeping gene, ACTIN2 transcripts was performed using 5′-GGTAACATTGTGCTCAGTGGTGG-3′ and 5′-GGTGCAACGACCTTAATCTTCAT-3′ primers. A final concentration of 200 nM primer was applied to the reaction mixture. The amplification program used for both ACTIN2 and IFN-α2a was 10 min of initial denaturation at 95 °C, followed by 40 cycles each of 15 s denaturation at 95 °C and 1 min of annealing and extension at 60 °C.

Quantification of miR-497 mRNA expression was conducted using a miScript SYBR^® Green PCR Kit (200) (Cat. No. 218073; Qiagen, Germany) according to the manufacturer’s guide and the endogenous control, GAPDH, was used for data normalization and relative quantification. The reaction was performed on the Step One qRT-PCR system (Applied Biosystems, CA, USA).
The primer sequence of miR-497 was F: 5′-ACACTGTGGTTTGTACGGCA-3′ and R: 5′-CTCCCCCACCCTCGCTCTAA-3′ and for the reference gene (GAPDH): F: 5′-GAC TCA TGA CCA CAGTCCATGC-3′ and R: 5′-AGA GGC AGG GAT GATGTT CTG-3′.
miR-497 relative mRNA expression was measured on the basis of the Cycle Threshold (CT) and normalized with GAPDH expression with the comparative formula 2^−ΔCt where {ΔCt = Ct (miR-497) − Ct (GAPDH)}.

Synthesized DHFR and sfGFP containing radioactive [³⁵S]Met were analyzed by 15% SDS-PAGE, and the gel image was visualized using a BAS-5000 bio-imaging analyzer (GE Healthcare, USA). Time-course analysis of sfGFP expression was performed by measuring sfGFP fluorescence every 3 min using a StepOne qRT-PCR system (#4376373, Applied Biosystems, USA). Fluorescence images of synthesized sfGFP in reaction mixtures were obtained using an LAS-4000 instrument (GE Healthcare, USA). The activity of synthesized DHFR was measured as described previously^{44 (link)}. Reaction mixtures (2 mL) contained 50 mM MES-KOH pH 7.0, 25 mM TRIS-HCl pH 7.0, 25 mM ethanolamine, 100 mM NaCl, 10 mM 2-mercaptoethanol, 0.1 mM EDTA, 100 μM dihydrofolic acid, and a 10 μL aliquot of the PURE reaction mixture, and they were incubated at 37°C for 15 min. Next, 20 mM NADPH was added to a final concentration of 200 μM, and the decrease in absorbance at 340 nm was measured over a period of 10 min with a V-550 spectrophotometer (Jasco, Japan). One unit of DHFR was defined as the amount of enzyme required to process 1 μmol of dihydrofolic acid in 1 min at 37 °C.