M mlv superscript 2

Total RNA was extracted from homogenized Stevia leaves using the TRIzol reagent (Invitrogen) and then treated with deoxyribonuclease I (DNase I; Roche, USA) to avoid possible genomic DNA contamination. Total RNA concentration was measured using a Nanodrop spectrophotometer, ND-1000 (Thermo Fisher Scientific, USA). One μg of total RNA was used for cDNA synthesis with M-MLV Superscript II (Promega, USA).
qRT-PCR was performed using SYBR Premix Ex Taq II (Takara, Japan) on the synthesized cDNA. The gene-specific primers are listed in Additional file 9: Table S1. The expression levels were quantified on Applied Biosystems (USA) 7900HT fast real-time PCR system. Stevia actin gene was used as an internal control for normalization. Specificity of the amplified PCR products was verified by regular PCR analysis and melting curve analysis on the qRT-PCR system. Biological and technical triplicates were carried out for each experiment.

Total RNA was isolated from plant samples using TRIzol reagent (Invitrogen, Carlsbad, CA USA) in accordance with the manufacturer’s instructions. cDNA was synthesized with 1 μg total RNA using MMLV Superscript II (Promega, Madison, WI, USA) after DNase I treatment (Roche Applied Science, Mannheim, Germany). Quantitative real-time PCR assay was performed on an ABI 7900 sequence Detection System (Applied Biosystems, Foster City, CA, USA). We used power SYBR Green PCR Master Mix (Applied Biosystems), using the manufacturer’s reagent protocol, but reducing the volume to 10 μl per reaction. As controls, we used the species-specific tubulin and actin primer sets for J. curcas and A. thaliana, respectively. Fold change values of the target gene transcripts were subsequently normalized by dividing the ^∆Ct values by the ^∆Ct values of each control gene transcript. All real-time PCR experiments were performed in triplicate using different biological samples. Sequences of primers used in PCR procedures are listed in Additional file 7.

Total RNA was extracted from homogenized Stevia leaves using TRIzol reagent (Invitrogen) and contamination from DNA was removed after treatment with deoxyribonuclease I (DNase I; Roche). For cDNA synthesis, 1 μg of total RNA was used with M‐MLV Superscript II (Promega, Madison, WI). To determine the transcript abundance of SrUGT76G1 and all other genes in the SGs biosynthesis pathway, qRT‐PCR was performed using SYBR Premix Ex Taq II (Takara, Shiga, Japan) and quantified on Applied Biosystems (USA) 7900HT fast real‐time PCR system. Primers used are listed in Table S1. Primer specificity was verified by sequencing of product from regular PCR and melting curve analysis. The abundance of Stevia actin transcript was used as an internal control for normalization.