Revertra ace rt master mix

Total RNA was extracted from tissues using an RNeasy ® Mini Kit and an RNase-Free DNase set (QIAGEN) as per the manufacturer's instructions. Complementary DNA was synthesized from purified RNA using ReverTra Ace® RT Master Mix (TOYOBO, Osaka, Japan) as per the manufacturer's instructions. Expression of genes GAPDH, IL-4, IL-6, IL-10, IFN-γ and TGF-β was evaluated by real-time PCR using a StepOnePlus TM Real-time PCR system and TaqMan® Real-time PCR Master Mix (Life Technologies). GAPDH was amplified as a control house-keeping gene. For all genes, pre-designed, commercially available primers and probes (Single Tube TaqMan® Gene Expression Assays: Life Technologies) were used. All real-time PCR assays were conducted by the StepOnePlus TM Real-time PCR system under the following conditions: 40 cycles of denaturation at 95 °C for 20 s, amplification at 95 °C for 1 s and quantification at 60 °C for 20 s. Gene expression level was calculated using the comparative Ct method as described elsewhere [21] .

RNAiso (TaKaRa) and ReverTra Ace RT master mix (Toyobo) were used for RNA extraction and reverse transcription, respectively. ExTaq was used for labeled PCR, and 18-24 PCR cycles were used to adjust for differences in transfection efficiencies. To detect spliced mRNAs, fragments were amplified using the sense primer A (ex17-up: 5′-CAACAAGAAACCACTGGCAC-3′) and the antisense primer (pFD-as-IRD1: 5′-GCCCAGCTTCACAGTAGTG-3′-IRD800), and the amplicons were electrophoresed through 2% agarose gel. To detect and quantify labeled PCR products, agarose gels were scanned using an Odyssey imager (Aloka). Applied volumes of PCR products were adjusted for the total intensity of each lane to appear even in the figures. To obtain data with higher resolution, PCR products generated using the sense primer B (ex2-sense: 5′-CAACAAGAAACCACTGGCAC-3′) and pFD-as-IRD1 were electrophoresed on a denaturing polyacrylamide gel using a dNA Sequencer LONG READIR 4200 (Aloka) according to the manufacturer's instruction. After gel electrophoresis, larger original scanned images were cropped using Image analysis (Aloka) and saved as TIFF files to transfer the files to another PC.

A rosette or cauline leaf was sampled and RNA was extracted from the sample using an RNeasy Plant Mini Kit (QIAGEN). RT reactions were performed using ReverTra Ace RT master mix (TOYOBO). For qRT-PCR, the reaction volume of 10 µL contained 5 µL of Thunberbird TM SYBR ® qPCR Mix (TOYOBO), and 0.2 µmol/L each of the primer pair, 1x ROX, and an aliquot of the template.
The real-time PCR reactions were performed with an ABI PRISM 7900 (Applied Biosystems) according to the following step-cycle program: pre-incubation at 90ºC for 10 min, followed by 40 cycles consisting of denaturation at 95ºC for 0.5 min, and annealing and extension at 60ºC for 1 min each. We used two different types of transgene-specific primer pairs, i.e., the LAC primer pair 5'-CCGCGGACCTCTCTGTTATC -3' & 5'-TGAACGTGTAGTTGGGGTCG -3' and the CBD prime pair 5'-TGACCATGCTGGTGCATTAT -3' & 5'-ATGTTGGGCTTGCTGTTTCT -3', plus a primer pair for the internal control gene,, i.e., the ACT2 primer pair 5'-TGATGCACTTGTGTGTGACAA -3' & 5'-ACAATGGGACTAAAACGCAAA -3'. The transgene expressions were calculated as the ratio of the quantity of transgene to that of ACT2.