M mulv reverse transcriptase

The tissue fragments of head kidney (HK) that were collected from the dissected fish were immediately preserved at −80 °C in TRIzol reagent (Thermo Fisher Scientific, San Jose, CA, USA) for RNA extraction. Total RNA was extracted from 0.5 g of olive flounder tissue using TRIzol reagent (Thermo Fisher Scientific, San Jose, CA, USA). Moreover, fish tissues were analyzed after quantitative analysis and purity assessment using a microvolume UV-Vis spectrophotometer (NanoDrop One, Thermo Fisher Scientific, San Jose, CA, USA). Then, the DNase I enzyme (Cosmogenetech, Seoul, Rep. of Korea) was mixed with the isolated RNA from tissues to exclude genomic DNA from the samples. Afterwards, we used the M-MuLV reverse transcriptase (Cosmogenetech) to produce complementary DNA (cDNA) from the samples. The qRT-PCR analyzer (Bio-Rad CFX96, Bio-Rad, Hercules, CA, USA) was then run with SYBR-Green reagent to determine the expression levels of the four selected immune-related genes such as FGH (flounder growth hormone), IL-1β (interleukin 1β), IL-10 (Interleukin 10), and β-actin (beta-actin as house-keeping gene) which were calculated by using CFX software in triplicates (CFX manager software version 2.0, Bio-Rad) (Table 2).

Tissue fragments from hexokinase (HK) were obtained and immediately stored at −80 °C in TRIzol reagent (Thermo Fisher Scientific, San Jose, CA, USA) for RNA extraction. Total RNA was extracted from 0.5 g of olive flounder tissue using TRIzol reagent (Thermo Fisher Scientific). Afterwards, it was quantified and the purity was assessed spectrophotometrically. The RNA was then treated with DNase I (Cosmogenetech, Seoul, Republic of Korea) to remove genomic DNA contamination. Complementary DNA (cDNA) was synthesized using M-MuLV reverse transcriptase (Cosmogenetech). The expression of four selected immune-relevant genes, including FGH (flounder growth hormone), IL-1β (interleukin 1β), IL-10 (interleukin 10), and β-actin (beta-actin as house-keeping gene), were analyzed via real-time PCR, which was performed using a Bio-Rad CFX96 (Bio-Rad, Hercules, CA, USA) with SYBR Green PCR Core Reagents (Cosmogenetech). The relative expression levels of the target gene transcripts (FGH, IL-1B, IL-10), with β-actin as an internal control, were calculated using CFX manager software version 2.0 (Bio-Rad) (Table 3). In all cases, each PCR was performed with triplicate samples.