U 0080d spectrophotometer

Melting points were determined using an MP-S3 apparatus (Yanaco, Tokyo, Japan). UV spectra were recorded at room temperature on a U-0080-D spectrophotometer (Hitachi, Tokyo, Japan). IR spectra were obtained with a FT-IR Spectrum RX I spectrophotometer (PerkinElmer, Waltham, MA, USA). Optical rotations were measured using a P-2000 digital polarimeter (JASCO, Tokyo, Japan). ¹H- and ¹³C-NMR spectra were recorded on Avance III HD 700 and Avance III 400 NMR spectrometers (Bruker, Billerica, MA, USA). Chemical shifts are shown in δ values (ppm) with tetramethylsilane as an internal standard. The ESIMS and HRESIMS were taken on a Bruker Daltonics APEX II 30e spectrometer (positive-ion mode). Column chromatography (CC) was performed on silica (70–230 mesh and 230–400 mesh, Merck, Darmstadt, Germany), Diaion HP-20 (Mitsubishi, Tokyo, Japan), and C₁₈ (Sigma-Aldrich, St. Louis, MO, USA) gels, respectively, and preparative TLC (thin-layer chromatography) was conducted on Merck precoated silica gel 60 F254 plates, using UV light to visualize the spots. High-performance liquid chromatography (HPLC) was performed on an LC-20AT series pumping system (Shimadzu, Kyoto, Japan) equipped with a Shimadzu SPD-20A UV-vis detector, and a SIL-10AF auto-sampling system at ambient temperature.

Total RNA was extracted from approximately 4 g of frozen tissues using the Large-Scale Column Plant RNAout kit (Tiandz, Inc., Beijing, China). To eliminate the remaining genomic DNA, the RNA was treated with DNase I (Takara, Dalian, China) according to the manufacturer's instructions. The integrity of the total RNA was assessed by running a 2 μl aliquot of the RNA sample on 1% formaldehyde denaturing agarose gel with ethidium bromide (EtBr). The concentration of RNA was estimated using a U-0080D spectrophotometer (Hitachi, Tokyo, Japan). Poly (A)⁺ RNA was isolated from 500 μg of total RNA with PolyATtract mRNA Isolation Systems (Promega, Madison, WI, USA). Double-stranded (ds) cDNA was synthesized from poly (A)⁺ using the SMART™ cDNA Library Construction Kit (Clontech, USA) according to the manufacturer's instructions and then purified using the QIAquick PCR Purification Kit (QIAGEN, Germany).