Stability of Reference Genes in Different Tissues of Triploid Rainbow Trout Oncorhynchus mykiss by Quantitative Real-Time PCR
SU Xiaoyan1,2, HAN Buying2, MENG Yuqiong1, BAI Xiaoyi1, LI Changzhong1, MA Rui2
1. College of Eco-Environmental Engineering, Qinghai University, Xining 810016, China; 2. State Key Laboratory of Plateau Ecology and Agriculture, Xining 810016, China
Abstract：Five candidate internal reference genes including gapdh (glyceraldehydes-3-phosphate dehydrogenase), ribosomal protein genes 2(rplp2), elongation factors1-α(ef1-α), beta-actin (β-actin), and 18S ribosomal RNA(18S rRNA) were comparatively expressed in brain, eyes, gills, skin, heart, kidney (head kidney, mid-kidney, and metanephros), liver, spleen, stomach, pyloric caeca, intestine (foregut, midgut, and hindgut) and muscle (red muscle, white muscle, and intermuscular compartment) of triploid rainbow trout Oncorhynchus mykiss with body weight of about 800 g by Real-time fluorescent quantitative PCR (qRT-PCR) method to screen stable reference genes in different tissues of triploid rainbow trout. The expression stability of the 5 candidate internal reference genes were analyzed by comparing the cycle threshold (Ct value) of the internal reference gene expression, and by three softwares including GeNorm, Best-Keeper, and NormFinder. The GeNorm analysis showed that the descending order of the M value was described as β-actin=rplp2<ef1-α<18S rRNA<gapdh<β-actin, with the minimum M value (1.42) of rplp2 genes. The Best-Keeper analysis revealed that the descending order of the standard deviation (SD) values was expressed as rplp2<ef1-α<β-actin<18S rRNA<gapdh and the coefficient of variation (CV) as rplp2<ef1-α<β-actin <gapdh < 18S rRNA, with the standard deviation (0.92) and coefficient of variation (3.94%) in rplp2 gene. The NormFinder analysis showed that the descending order of the average stable expression value of the five candidate reference genes were listed as rplp2=β-actin<ef1-α<18S rRNA<gapdh, β-actin, with the minimal stable value (0.66) in rplp2 genes. In conclusion, rplp2 and β-actin genes had the maximal expressional stability in different tissues of triploid rainbow trout, and can be used as reference gene for qRT-PCR.
苏晓燕, 韩步鹰, 孟玉琼, 白晓易, 李长忠, 马睿. 三倍体虹鳟实时荧光定量PCR内参基因稳定性分析[J]. 水产科学, 2022, 41(1): 35-43.
SU Xiaoyan, HAN Buying, MENG Yuqiong, BAI Xiaoyi, LI Changzhong, MA Rui. Stability of Reference Genes in Different Tissues of Triploid Rainbow Trout Oncorhynchus mykiss by Quantitative Real-Time PCR. 水产科学, 2022, 41(1): 35-43.
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2022, Vol. 41 
