Primescript 2 rtase

The cDNA was synthesized from the flowering stage of the Welsh onion with total RNA in a 10-µL reaction system using PrimeScript II RTase (TaKaRa Biotechnology, Dalian, Liaoning Province, China). The reverse transcription reaction mixture contained 5 µL of total RNA (0.8 µg), 3 µL of diethylpyrocarbonate (DEPC) water, 1 µL of oligo dT (50 µM) and 1 µL of dNTP (10 mM), and the reaction was performed following the kit protocol. Three biological replicates and three technical replicates for each experiment were performed. The qPCR reactions were performed in 96-well plates on the LightCycler 480 instrument (Roche Diagnostic, Mannheim, Germany) using SYBR Green I (TaKaRa Biotechnology, Dalian, Liaoning Province, China). The qPCR primers, which were designed using Primer3 (http://frodo.wi.mit.edu/primer3/), are shown in Table S3. Each gene was analyzed using the method described previously by Qianchun Liu et al. [1 (link)].

Total RNA was extracted from cells using the RNeasy mini kit with QIAshredder (Qiagen, Venlo, The Netherlands) following the manufacturer’s instructions. cDNA was prepared using Primescript II RTase (Takara, Otsu, Japan) and random primers. PCR reaction was performed using Takara ExTaq HS (Takara). Relative quantification analyses (qRT-PCR) were performed using a MX3000P system (Agilent, Santa Clara, CA, USA) using SYBR Green dye chemistry. The amount of 18S rRNA was measured as an internal control for qRT-PCR. All primers are listed in Table S1.

Total RNA was extracted from the roots of control S. baicalensis seedlings and those of seedlings treated with 200 µM MeJA for 0, 1, 3, or 7 h using TRIzol Reagent. Total RNA samples was then reverse-transcribed to first-strand cDNA using PrimeScript II RTase (Takara cat. no. 6110A). Primer pairs were designed using Primer Premier 5 software. Quantitative PCR was performed on a QuantStudio 3 System following the manufacturer’s protocol. The relative expression levels of target genes were calculated using the 2^–ΔΔCt method with SbACT7 as the reference gene. The primer sequences are available in Table S1.

Total RNA was extracted from cells or Exo using TRIzol (Ambion, USA). RNA was then reverse transcribed to cDNA using PrimeScript II RTase (TAKARA, Japan). The cDNA was amplified using SYBR FAST qPCR Master Mix (KAPA biosystems). Reaction conditions were 40 cycles of 95°C for 3 min, 95°C for 5 s, and then 56°C for 10 s followed by 72°C for 25 s. The primer sequences are listed in Table 1. GAPDH and U6 gene were used as the housekeeping gene. The mRNA was calculated using the 2^-ΔΔt method.

The cDNA was synthesized from total RNA using PrimeScript II RTase (TaKaRa, Japan) in a 10 µL reaction system. The reverse transcription reaction mixture contained 5 µL of total RNA (0.8 µg), 1 µL of oligo dT (50 µM) (TaKaRa, Japan), 1 µL of 10 mM of each dNTP and 3 µL of DEPC water. The mixture was incubated at 65°C for 5 min and cooled on ice for 5 min, after which 4 µL of 5× PrimeScript II Buffer, 0.5 µL of RNase Inhibitor (40 U/µL), 1 µL of PrimeScript II RTase (200 U/µL) and 4.5 µL of DEPC water were added. The mixture was incubated at 45°C for 1 h. The enzyme was inactivated by incubating at 95°C for 5 min. The qPCR was carried out on the LightCycler 480 II Real-Time PCR Detection System (Bio-Rad, USA) with SYBR Green I (TaKaRa, Japan). The primers used in the qPCR to validate the differentially expressed genes are shown in Table S2. Each gene was analyzed in triplicate, after which the average threshold cycle (CT) was calculated per sample, and an endogenous actin gene was used for normalization. The relative fold changes in gene expression were calculated using the comparative Ct (2^−ΔΔCt) method.

The following is a list of the main materials and reagents used in this study: OVA (A5253, sigma, Ronkonkoma, NY, USA); BCA Protein Concentration Assay Kit (PC0020, solarbio, Beijing, China); Oligo (dT)18/miR-RT Primer (3806, TAKARA, Kusatsu, Japan); PrimeScript II Rtase (2690A, TAKARA, Japan); T4 DNA ligase (Fermentas, Waltham, MA, USA); Dual luciferase reporter gene assay kit (RG027, Beyotime, Shanghai, China); Masson Trichrome Stain Kit (G1340, Solarbio, Beijing, China); ELISA kits IL-4 (HM10380), IL-5 (HM10213), IL-13 (HM10191) and IFN-γ (HM10058), purchased from Bioswamp(Wuhan, China); DAB Concentrate Kit (DA1010, Solarbio, Beijing, China); AnnexinV-PE/7 AAD Apoptosis Assay Kit (559763, BD, Becton Drive Franklin Lakes, NJ, USA).

Total RNA was isolated using ISOGEN II (Nippon Gene) and NucleoSpin RNA (Macherey-Nagel). cDNA was generated with PrimeScript II RTase (Takara) or ReverTra Ace qPCR RT Master Mix (Toyobo). Semi-qPCR was performed with EmeraldAmp MAX PCR Master Mix (Takara). qPCR was performed with PowerUp SYBR Green Master Mix (Thermo) using the StepOne system (Thermo). Copy numbers were determined using standard curves from pEGFP-P4-ATPases, pMSCs and pEGFP-AP3D1, and compared to Ap3d1⁶⁴ . Relative expression was calculated using the 2^−ΔΔCt method. Primers are listed in Supplementary Tables 3 and 4.