Takara primescript 2 1st strand cdna synthesis kit

Total RNA was extracted from cell samples using TaKaRa MiniBEST Universal RNA Extraction Kit (TaKaRa, Tokyo, Japan) and was converted into complementary DNA (cDNA) using TaKaRa PrimeScript II 1^st Strand cDNA Synthesis Kit (TaKaRa, Tokyo, Japan). Small RNAs were isolated using miRcute miRNA isolation kit (Tiangen Biotech, Beijing, China) and converted into cDNA using Mir-X^TM miRNA First-Strand Synthesis Kit (TaKaRa, Tokyo, Japan) or with specific primers. Quantitative polymerase chain reaction (qPCR) was performed using QuantiNova SYBR Green PCR Kit (QIAGEN, Hilden, Germany). The protocol of real-time qPCR was as follows: initially 95°C for 2 min, followed by 40 cycles of 95°C for 5 s and 60°C for 15 s. The sequences of the primers used are displayed in Table 1. Quantifications were normalized by taking GAPDH or U6 as an endogenous control and were calculated by the 2^−ΔΔCt method and purchased from TsingKe Biological Technology (Beijing, China). All experiments were repeated in triplicate.

Total RNA was extracted from the leaf tissues of the fine-stem stylo variety ‘YueNong 01′ using TransZol Plant kit (TransGen Biotech, Beijing, China). The first cDNA was synthesized using TaKaRa PrimeScript II 1st Strand cDNA Synthesis Kit (Takara Biotechnology, Shiga, Japan). The full-length coding DNA sequences (CDSs) of SgGH3.1 were amplified by PCR using a specially designed primer pair, SgGH3.1-pBA-F and SgGH3.1-pBA-R (Table S1). PrimeSTAR^® Max DNA Polymerase (Takara Biotechnology, Japan) was used for the PCR. The 50 µL PCR reaction system contained 25 µL PrimeSTAR Max Premix (2X), 0.2 µM SgGH3.1-pBA-F, 0.2 µM SgGH3.1-pBA-R, and approximately 200 ng cDNA template. The PCR protocol consisted of 30 cycles of 20 s at 98 °C, 5 s at 55 °C and 30 s at 72 °C. The amino acid sequence of SgGH3.1 was aligned with 20 Arabidopsis GH3 proteins (Table S2) and 14 leguminous GH3.1 proteins (Table S3) using T-coffee [20 (link)]. A phylogenetic tree was constructed from the alignment using MrBayes [21 (link),22 (link)]. Percentage identities between the leguminous GH3.1 proteins were calculated based on the alignment.

For the isolation of total RNA from OPCs, TRIZOL Reagent (Thermo Fisher Scientific) was used. Takara PrimeScript II 1st strand cDNA synthesis kit (Takara-Bio) was used for cDNA synthesis obeying supplier’s instructions. 20 μl of qRT-PCR reaction mixture consisted of 10 μl of BIO-RAD iQ SYBR Green Supermix (BIO-RAD), 5 μl of primer mix (2 μM of each forward and reverse primers) and 5 μl of template cDNA (0.2 ng/μl). The sequences of PCR primers for qRT-PCR are shown in Supplementary Table S3. Real-time PCR was performed on a CFX96 Real-Time System (BIO-RAD). The condition of PCR was 5 seconds at 95 °C for the denaturing, 30 seconds at 60 °C for the annealing and the extension. The total numbers of cycling were 40. The resulting values were normalized to the endogenous control gene, glyceraldehyde 3-phosphate dehydrogenase (Gapdh) or β-actin (Actb). Results were presented as the relative expression to that of the control using the comparative Ct method (∆∆Ct).

Fifteen days after transplanted to soil, transgenic and wild-type (WT) tobacco leaf surface was painted with 0.2% (W/V) basta. One week after the basta treatment, plantlets with no leaf injury were selected for further investigation. DNA was isolated from fresh leaves using Plant Genomic DNA Kit (TianGen Biotech, China) and amplified with two primer pairs, bar-F and bar-R, SgRVE6-F1 and SgRVE6-R1 (Supplementary Table 1). Total RNA was isolated from fresh leaves using RNAprep Pure Plant Kit (TianGen Biotech, China). The first cDNA was synthesized using TaKaRa PrimeScript II 1st Strand cDNA Synthesis Kit (Takara Biotechnology, Japan) and used for PCR amplification with primer pair, SgRVE6-F2 and SgRVE6-R2 (Supplementary Table 1). The WT plants served as a negative control and pBA002-SgRVE6 plasmid carrying bar gene and SgRVE6 as a positive control. The transgenic and WT tobacco plants were cultivated at 25 °C and 70% humidity under a 16/8-h (light/dark) photoperiod with a photon flux density (PFD) of 60 μmol m⁻² s⁻¹.

Ten fine-stem stylo plants were vegetatively propagated by cuttings. Fresh shoots of 10 cm were cut from a single fine-stem stylo plant and cultured in water for rooting. After one week, rooted shoots were planted in pots (10 cm in height and 12 cm in diameter) containing soil mixtures of Jiffy^® substrate and vermiculite (3:1) and cultured in a growth chamber at 28°C and 70% humidity under a 16/8-h (light/dark) photoperiod for four weeks. Plants of uniform growth were selected for cold treatment at 4°C and 70% humidity under a 16/8-h (light/dark) photoperiod. Root, leaf and stem tissues were harvested at 0, 2, 6, and 12 h after cold treatment. Total RNA was extracted from nitrogen-frozen tissues using TransZol Plant Kit (TransGen Biotech, China). First-strand cDNA was synthesized using TaKaRa PrimeScript II 1st Strand cDNA Synthesis Kit (Takara Biotechnology, Japan). The synthesized cDNA was used as template for Real-time PCR in a CFX-96 Real-time System (BioRad, USA) with the specific primer pair, SgRVE6-F1 and SgRVE6-R1 (Supplementary Table 1). Real-time PCR was conducted using SYBR Premix Ex Taq II kit (Takara Biotechnology, Japan). The Real-time PCR reaction solution and procedure followed the manufacturer’s instruction. The results were analyzed by Bio-Rad CFX Manager Software 1.6.

RNA was extracted from kidney and liver using Qiacube and the RNeasy Mini Kit (74106; Quiagen, Venlo, The Netherlands) as described previously [28 (link),33 (link)]. cDNA was synthesized using a Takara PrimeScript II 1st strand cDNA Synthesis Kit (6210; Takara Bio). Real-time PCR was performed using probes form Applied Biosystems, Waltham, MA, USA (β-actin Rn00667869_m1, Epo Rn00667869_m1, Hif2α Rn00576515_m1, Hif1α Rn01472831_m1, Phd2 Rn00710295_m1, Gr (Nr3c1) Rn00561369_m1, Rhcg Rn00788284_m1), and Premix Ex Taq (RP39LR; Takara Bio). mRNA expressions in control and IRI rats were compared by relative gene expression data using real-time quantitative PCR and the 2^−ΔΔCT.

Total RNA was extracted from the fresh leaf of the fine-stem stylo plants using TransZol Plant Kit (TransGen Biotech, China). The first cDNA was synthesized using TaKaRa PrimeScript II 1st Strand cDNA Synthesis Kit (Takara Biotechnology, Japan). The full-length coding DNA sequence (CDS) of SgRVE6 was amplified using the primer pair SgRVE6-F and SgRVE6-R (Supplementary Table 1). The primers were designed based on SgRVE6 transcript sequence from our previous transcriptome assembly. The amino acid sequences of SgRVE6 was aligned with 14 leguminous RVE6s (Supplementary Table 2) and the 11 members of the RVE family in A. thaliana (Supplementary Table 3) using T-Coffee. Phylogenetic trees were constructed from the alignment by Unipro UGENE1.27 using MrBayes method. The physicochemical property of SgRVE6 was analyzed by ProtParam (http://web.expasy.org/protparam/).