Selection of reference genes for quantitative real‐time PCR normalisation in largemouth bass Micropterus salmoides fed on alternative diets

Abstract

The partial cDNA sequences of eight reference genes (actb, tuba1, gapdh58, gapdh59, eef1a1, RNA 18 s, pabpc1, ube2I) were cloned from largemouth bass Micropterus salmoides. The expression levels of these eight genes were compared in the various tissues (eye, spleen, kidney, gill, muscle, brain, liver, heart, gut and gonad) of M. salmoides fed on forage fish. The results showed that the candidate genes exhibited tissue‐specific expression to various degrees and the stability ranking order was eef1a1 > tuba1 > RNA 18 s > pabpc1 > ube2I > actb > gapdh58 > gapdh59 among tissue types. Four candidate genes eef1a1, tuba1, RNA 18 s and actb were used to analyse the stability in liver tissues of largemouth bass between the forage‐fish group and the formulated‐feed group. The candidate genes also showed some changes in expression levels in the livers, while eef1a1 and tuba1 had the most stable expression in livers of fish fed on alternative diets within 10 candidates. So eef1a1 and tuba1 were recommended as optimal reference gene in quantitative real‐time PCR analysis to normalise the expression levels of target genes in tissues and lives of the M. salmoides fed on alternative diets. In livers, the expression levels of gck normalised by eef1a1 and tuba1 showed the significant up‐regulation in formulated feed group (P < 0.05) than those in forage‐fish group. While sex difference has no significant effects on the expression levels of gck in both groups.

Changes of DNA Methylation Pattern in Metabolic Pathways Induced by High-Carbohydrate Diet Contribute to Hyperglycemia and Fat Deposition in Grass Carp (Ctenopharyngodon idellus)

“…The temperature gradually increased from 65 to 95°C, with 0.5°C every 6 s. According to the instructions of GoTaq® qPCR Master Mix (Bio-Rad, USA), the reaction was composed of 0.2 μM of primers, 1 μl cDNA, 10 and μl GoTaq® qPCR Master Mix and supplied with double-distilled water at 20 μl. The mRNA expression levels were quantified relative to the expression of elongation factor 1 α ( ef1 -α) as endogenous reference ( 20 ) and analyzed by the optimized comparative Ct (2 ΔΔ Ct ) value method ( 21 ).…”

Selection of internal reference gene for quantitative real‐time PCR analysis of gene expression in largemouth bass ( Micropterus salmoides ) under different experimental conditions

“…Previous studies also indicated that an internal reference gene often has diverse expression levels in different species, different cells or even different experiment conditions. Thus, the selection of a reliable internal reference gene is one of the important factors to detect quantification of the target gene expression analysis by qPCR (Ma et al, 2019). The software of geNorm, NormFinder and BestKeeper can further calculate the values of reference gene expression stability and give comprehensive ranking of reference genes.…”

Comparative Transcriptomic Analysis of Largemouth Bass (Micropterus salmoides) Livers Reveals Response Mechanisms to High Temperatures

“…Quantitative PCR was carried out on a LightCycler ® 480 II instrument (Roche, Basel, Switzerland) using a TB Green ® Premix Ex Taq™ II Kit [48]. The relative mRNA expression levels were calculated using the 2 −∆∆CT method [49], and the β-actin gene of largemouth bass was used as an internal control [50]. The primers used for qRT-PCR are listed in Table S1.…”