Maxima h minus first strand cdna synthesis kit with dsdnase

Cells were incubated for 24 h with the seaweed extracts (dosages based on saringosterol content), RXR agonist bexarotene (1 µM; #153559-49-0, Merck), the LXRα/β agonist T0901317 (1 µM), PPARα agonist WY-14643 (50 µM), PPARγ agonist pioglitazone (10 µM), or the extract or compound solvent ethanol or DMSO. Cells were washed with cold phosphate-buffered saline and RNA was isolated using Trizol (Thermo Fisher Scientific) and reverse transcribed to cDNA using the Maxima H Minus First Strand cDNA Synthesis Kit with dsDNase (Thermo Fisher Scientific), according to the manufacturer’s instructions. Quantitative real-time PCR (qPCR) was conducted in duplicate with 10 ng cDNA on a CFX384 Thermal Cycler (Bio-Rad Laboratories) using the PowerTrack™ SYBR Green Master Mix (Applied Biosystems) and the following cycling conditions: 95 °C for 2 min and 40 cycles of [95 °C for 15 s, 60 °C for 60 s]. The intron-spanning primers for qPCR were designed with Primer-BLAST [60 (link)]. Primer sequences are listed in Table 1. Relative quantification of the gene expression was accomplished with the comparative Ct method. The data were normalized to five reference genes (ACTB, B2M, HPRT1, SDHA, and YWHAZ) and expressed as fold change relative to the EtOH or DMSO control. The experiments were performed three times.

In order to characterize coding sequences, total RNA from a pool of 10 snails was extracted with TRIzol reagent (ThermoFisher Scientific, Paris, France) according to the manufacturer’s instructions and subsequently reverse transcribed to first strand cDNA using Maxima H Minus First Strand cDNA Synthesis Kit with dsDNase (ThermoFisher Scientific, Paris, France).
Four Glabralysin gene fragments (Gla1Aa1, Gla1Aa2, Gla1Ba1, Gla1Ca1) were obtained by running polymerase chain reaction (PCR), using GoTaq G2 Hot Start enzyme (Promega, Lyon, France). Briefly, 2.4 µL MgCl₂ (1.5 mM), 8 µL Gotaq Buffer 5×, 0.8 µL dNTPs (10 mM), 0.2 µL Gotaq enzyme (1 unit), and 0.5 µL of primers (100 µM), for a total reaction volume of 40 µL. Primer sequences are shown in Table S1. The amplification cycling conditions were as follows: initial denaturation step at 95 °C for 6 min, followed by 40 amplification cycles composed of 30 s denaturation at 95 °C, 30 s primer annealing step at 51 °C and 1 min elongation at 72 °C. PCR reaction was ended by a final elongation step of 5 min at 72 °C. Amplicons were sequenced by Sanger method. Glabralysin gene structure (exon/intron) was determined by comparing cDNA sequences obtained from sequencing to the B. glabrata BglaB1.6 genome assembly using VectorBase website [49 ].