Easyspin plus total rna kit

Ramie Chuanzhu 8, which is moderately resistant to RM, was used in this study. Ramie seedlings were prepared with the shoot-cutting propagation method. The seedlings were cultured in a climate chamber at 26°C  ±  1°C, 75%  ±  1% relative humidity, with a photoperiod of 14 : 10 (L : D). RM egg masses attached to the back of ramie leaves were collected from infested ramie fields at the Institute of Bast Fiber Crops (112.11′E, 28.51′N). For hatching, the eggs were subjected to the same conditions as the ramie seedlings, and J2 larvae were used as inoculums. To prepare the challenged plants (CH), two J2 larvae were transferred to the ramie seedlings onto the fourth leaf from the shoot apex. To stimulate their appetite, larvae were kept on fasting for 12 h prior to inoculation. At 12, 24, 48, and 72 h after inoculation, the two topmost undamaged leaves were sampled. Unchallenged control (CK) plants were sampled simultaneously. There were five plants per treatment, and their sampled leaves were pooled per treatment. Overall, eight pooled samples were obtained (CK12, CK24, CK48, CK72, CH12, CH24, CH48, and CH72). Total RNA from each pooled sample was extracted using a commercially available EASYspin plus Total RNA kit (Aidlab, Beijing, China), following the manufacturer's protocol. The obtained RNA was subsequently stored at −80°C.

Root tissues of ramie in a 2 mL Eppendorf tube (pre-chilled in liquid nitrogen) were ground to fine powder using a Tissuelyser-24 Multi-Sample Tissue Grinder (Shanghai Jingxing, Shanghai, China). Total DNA and RNA were extracted using a Plant DNA Mini Kit and an EASYspin Plus Total RNA Kit (both from Aidlab, Beijing, China), following the manufacturer’s protocol. A Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) was used to measure DNA and RNA concentration, and first-strand cDNA samples were synthesized from about 1 μg of the total RNA using a RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific).