Rnaiso plus reagent rna extraction kit

Total RNA was extracted from the hepatopancreas, epidermis, muscle, midgut, and hindgut tissues using RNAiso™ plus reagent (RNA Extraction Kit, TaKaRa, Japan) according to the manufacturer's protocol. The concentration and quality of the total RNA were estimated using a micro-volume ultraviolet-visible spectrophotometer (Quawell Q5000; Thmorgan, China) and agarose-gel electrophoresis, respectively. The samples were reverse transcribed using the PrimeScript™ RT reagent Kit (Perfect Real Time, TaKaRa, Japan) according to the manufacturer's protocol. The obtained cDNA was diluted to 1:2 with double-distilled water and used as the qRT-PCR template. Relative quantification was performed using the ABI 7500 Real-Time PCR System (Life Technology, USA) with a SYBR® Premix Ex Taq™ (Tli RNaseH Plus, TaKaRa, Japan) kits using the following program: 95°C for 30 s; 40 cycles at 95°C for 5 s, 60°C for 34 s; followed by a melting curve at 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s. The PCR primer sequences for EcR, MIH, and Chi are presented in Table 1 (Sangon Biotech Co. Ltd., Shanghai, China). β-actin was used as the internal control and performed in triplicate for each sample. Relative changes in the level of gene expression were determined by the 2^−ΔΔCt method. Data were analyzed and presented as the average values ± standard deviation (SD).

Total RNA was extracted from E. sinensis using the RNAiso Plus reagent (RNA Extraction Kit, TaKaRa, Japan) according to the manufacturer’s instructions. Briefly, tissues were ground in a mortar with liquid nitrogen and collected in 1.5 ml centrifuge tubes. The RNAiso Plus reagent was added (1 ml), and samples were left at room temperature for 5 min. Samples were then centrifuged 5 min at 4°C and 12000 rpm, and the supernatant was collected in new 1.5 ml tubes. Chloroform (200 μl) was added, and samples were then oscillated and again left at room temperature for 5 min before a 15 min centrifugation at 4°C and 12000 rpm. The supernatant was collected in new 1.5 ml tubes and 500 μl of isopropyl alcohol was added. Samples were left at room temperature for 10 min and then centrifuged 10 min at 4°C and 12000 rpm. Pellets were washed with 1 ml of 75% alcohol and centrifuged 10 min at 4°C and 7500 rpm. The supernatant was removed, and the remaining pellets were dried and dissolved in 30 μl of DEPC-treated water. The concentration and quality of the total RNA were estimated by micro-volume ultraviolet-visible spectrophotometer (Quawell Q5000; Thmorgan, China) and agarose-gel electrophoresis, respectively.