Identification of reference genes for quantitative expression analysis in Indian catfish,Clarias magur, under physiological and pathological conditions

Abstract

The reverse transcription quantitative real‐time PCR (RT‐qPCR) is widely preferred tool for expression analysis of target gene at mRNA level. However, in order to obtain significant biological comparison, data normalization to a stable endogenous internal control gene also called reference gene (RG) is of utmost importance. In the present study, we evaluated the expression stability of four commonly used housekeeping genes, that is beta actin, β‐actin; glyceraldehyde‐3‐phosphate dehydrogenase, GAPDH; elongation factor‐1 alpha, EF‐1α and 18S ribosomal RNA, 18S for qPCR data normalization. This study was conducted for two varying physiological (Group I: fed vs. non‐fed and Group II: earthen pond vs. fibre reinforced plastic tank reared) and one pathological condition (Group III: Aeromonas hydrophila injected vs. PBS injected) in seven different tissues of Indian catfish, Clarias magur, an economically valuable food fish of South‐East Asia. Tissue wise expression stability analysis using web‐based comprehensive tool Reffinder revealed that EF‐1α was the most stable RG in kidney, liver, spleen and gills of group I; spleen, intestine, muscle and brain in group II; and kidney, liver, intestine, muscle and brain in group III. β‐actin was the most stable RG in intestine of group I; liver and brain in group II; spleen and gill in group III. 18S was the most stable RG in muscle and brain of group I; kidney and gill in group II. GAPDH was the least stable RG among four selected genes under all the experimental conditions. These findings on selection of RGs will lead to precise interpretation of relative gene expression in different tissues by RT‐qPCR in C. magur in the context of physiological studies and bacterial infection.

Transcriptome Profile Analyses of Head Kidney in Roach (Rutilus rutilus), Common Bream (Abramis brama) and Their Hybrids: Does Infection by Monogenean Parasites in Freshwater Fish Reveal Differences in Fish Vigour among Parental Species and Their Hybrids?

“…Because of no obvious differences in differentially expressed genes between some pairs of fish lines and because of high intra-line variability in P. homoion infection, the correlation between the relative expression changes upon infection for each fish individual (gene density) and P. homoion infection was calculated for each gene. Using a correlation coefficient (r > 0.65 and p ≤ 0.001), 10 genes with a relevant biological function (checked using published studies [ 88 , 89 , 90 , 91 , 92 , 93 , 94 , 95 , 96 , 97 , 98 , 99 , 100 , 101 ]) were selected for qPCR validation, 8 of them exhibiting a positive correlation and 2 of them a negative correlation with P. homoion infection. Two of these selected genes ( YQAJ and MAM ) were finally removed from the analyses because of unspecific PCR amplification.…”

Overcoming taxonomic challenges in DNA barcoding for improvement of identification and preservation of clariid catfish species

“…As of April 2023, public repositories have amassed 59,304 nucleotide sequences from clariid catfish, and they include 3,748 mitochondrial and 55,556 nuclear sequences. Most of the nuclear sequences were obtained from functional gene analyses [ 44 - 47 ]. Over 5.11% of these nucleotide sequence resources contain COI , Cytb , and D-loop sequences, which are popular markers for molecular taxonomy and species identification of clariid catfish [ 3 , 13 , 15 , 17 - 20 ].…”

“…As of April 2023, public repositories have amassed 59,304 nucleotide sequences from clariid catfish, and they include 3,748 mitochondrial and 55,556 nuclear sequences. Most of the nuclear sequences were obtained from functional gene analyses [44][45][46][47]. lecular taxonomy and species identification of clariid catfish [3,13,15,[17][18][19][20].…”