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| Selection of Reference Genes for Real-Time Quantitative PCR of Peanut Worm Sipuculus nudus |
| PENG Bingbing1, BIN Dongchao1, ZHOU Yuna2, YANG Zhihui3, CAI Xiaohui1, PENG Yinhui1 |
1. Guangxi Key Laboratory of Beibu Gulf Marine Biodiversity Conservation, Ocean College, Beibu Gulf University, Qinzhou 536011, China;
2. Beihai Fishery Technology Extension Station, Beihai 536000, China;
3. Xingtai Bureau of Agriculture and Rural Affairs, Xingtai 054000, China |
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Abstract At present, the main method to study the quantitative expression of genes is real-time quantitative PCR(qRT-PCR), and the accuracy of qRT-PCR analysis can be improved by selecting the appropriate internal reference genes. In this study, EF1-a2,a-Tub1, RPL13-b, Actin1, H3-b and GAPDH1 were analyzed by qRT-PCR from the rostrum, somatic muscles, renal duct, brain, intestine, adductor muscle, esophagus, and blood lymphocyte of peanut worm Sipunculus nudus, the expression of six candidate internal reference genes was stable, and the stability of internal reference genes was verified by IgGFc-binding protein. BestKeeper software revealed that the descending order of the stability of internal reference genes was expressed as:RPL13-b＞H3-b＞EF1-α2＞GAPDH1＞Actin1＞α-Tub1; Normfider software analysis selected the stability of the internal reference gene, with the descending order of RPL13-b＞EF1-α2＞GAPDH1＞H3-b1＞Actin1＞α-Tub1; The descending order of stability of internal reference genes selected by geNorm software analysis was described as: RPL13-b=H3-b＞EF1-α2＞GAPDH1＞Actin1＞α-Tub1. When the stable internal reference gene RPL13-b was used to calibrate the qRT-PCR data, the expression trend of IgGFc-binding protein gene was basically the same as in different tissues. The most unstable internal reference gene-Tub1 was used to calibrate the qRT-PCR data, with different change trend of expression level of IgGFc-binding protein gene. The expression of RPL13-b internal reference gene was the most stable in the whole tissues of peanut worm and was used as the optimal single internal reference gene.
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Received: 18 May 2020
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