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| 黄带拟鲹qRT-PCR内参基因筛选及验证 |
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李杰锋1,2, 王焕2, 李步苏2,3, 曾祥辉2,4, 柳淑芳2,3, 庄志猛2
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1.上海海洋大学水产与生命学院 上海 201306;2.中国水产科学研究院黄海水产研究所 农业农村部
海洋渔业与可持续发展重点实验室 山东 青岛 266071;3.海洋渔业科学与食物产出过程
功能实验室 山东 青岛 266071;4.大连海洋大学水产与生命学院 辽宁 大连 116023
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| 摘要: |
| 实时荧光定量PCR (qRT-PCR)是研究基因表达的一种广泛使用且有效的方法，选用黄带拟鲹(Pseudocaranx dentex)合适的内参基因，是qRT-PCR技术获得该物种基因表达可靠结果的关键。本研究首先测定了9个常用内参基因(β-actin、RPL13、EF-1α、GAPDH、HPRT、PPIA、β2M、TUB和PP2A)在黄带拟鲹成鱼组织中的表达丰度；同时，利用BestKeeper、NormFinder、geNorm和RefFinder 4种模型预测了内参基因的表达稳定性，发现其表达稳定性排序为RPL13 >EF-1α > PP2A >HPRT >PPIA >TUB >β2M >β-actin >GAPDH。进一步通过定量检测目标基因myod1在不同组织的表达情况，验证上述预测结果的准确性。研究发现，RPL13和EF-1α单独或联合作为黄带拟鲹qRT-PCR内参基因，可显著提高目标基因表达量检测结果的稳定性与可靠性。结果表明，RPL13和EF-1α可作为黄带拟鲹不同组织qRT-PCR分析的内参基因。同时，也证实了并不是管家基因的表达在所有物种中都具有良好的稳定性，需要根据实际情况筛选适宜的内参基因。本研究结果可为后续黄带拟鲹功能基因表达特征的研究提供技术支撑，有望适用于其他鲹科鱼类。 |
| 关键词: 黄带拟鲹 qRT-PCR 内参基因 稳定性 |
| DOI：10.19663/j.issn2095-9869.20220622001 |
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| Screening and evaluating reference genes for quantitative real-time PCR in striped jack (Pseudocaranx dentex) |
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LI Jiefeng1,2, WANG Huan2, LI Busu2,3, ZENG Xianghui2,4, LIU Shufang2,3, ZHUANG Zhimeng2
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1.College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China;2.Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of Marine Fisheries and Sustainable Development, Ministry of Agriculture and Rural Affairs, Qingdao 266071, China;3.Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao 266071, China;4.College of Fisheries and Life Science, Dalian Ocean University, Dalian 116023, China
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| Abstract: |
| Quantitative real-time polymerase chain reaction (qRT-PCR) is one of the most widely used molecular techniques, often implemented to allow for the detection and quantitation of gene expression because of its high sensitivity, specificity, and reproducibility. However, reliable and comparable relative quantitative results using qRT-PCR require the application of appropriate reference genes in order to eliminate non-biological variations caused by initial RNA templates, efficiency of cDNA synthesis, and laboratory procedures. However, previous studies have reported that the stability of many reference genes may vary across species, tissue types, cell lines, developmental stages, and experimental treatments, yielding inaccurate or incorrect gene expression results. Therefore, the selection and validation of stable reference genes for different tissues from a specific species are especially important for obtaining accurate target gene expression results. The striped jack, Pseudocaranx dentex, belonging to the order Perciformes and family Carangidae, is a pelagic migratory fish with high nutritional value. This fish has already received extensive attention in global aquaculture production and is regarded as a candidate species for far-reaching marine aquaculture in China. Given this, there are currently a large number of fairly extensive molecular biology and genetics studies of P. dentex underway, which in turn have increased the demand for quantitative gene expression analysis by qRT-PCR in these animals. However, few studies have evaluated the reference genes for this species. Thus, the objective of this study was to identify suitable reference genes in different tissues of P. dentex, in an effort to provide the necessary tools to support subsequent gene expression pattern analysis.
We evaluated nine commonly used reference genes, including beta actin (β-actin), ribosomal protein L13 (RPL13), elongation factor 1 alpha (EF-1α), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine phosphoribosyl transferase (HPRT), peptidylprolyl isomerase A (PPIA), beta 2-microglobulin (β2M), beta tubulin (TUB), and serine/threonine-protein phosphatase 2A catalytic subunit (PP2A) using qRT-PCR analysis across various P. dentex tissues. These evaluations included the study of their expression stability across ten tissues, including the brain, gill, heart, intestine, kidney, liver, spleen, stomach, slow-twitch muscle, and fast-twitch muscle, from three adult individuals of P. dentex using four independent methods, namely BestKeeper, NormFinder, geNorm, and RefFinder. These results were then validated in the qRT-PCR profiling of the myoblast determination protein 1 (myod1) gene in both muscle (slow-twitch and fast-twitch muscles) and non-muscle tissues (kidney and gills) using the various recommended reference genes or their combinations.
Expression analysis showed that RPL13 was the most highly expressed gene in these samples, followed by EF-1α, β2M, β-actin, PPIA, HPRT, TUB, and PP2A, whereas GAPDH was the most weakly expressed across all ten P. dentex tissues. In addition, all nine candidate reference genes exhibited relatively inconsistent variations in Ct value across various tissues, with the BestKeeper stability assay, which uses standard deviation (SD) and coefficient of variation (CV) to score the candidates, suggesting that these genes could be ordered from most stable to least stable as follows: EF-1α > RPL13 > PP2A > PPIA >HPRT > β2M > TUB > GAPDH > β-actin. NormFinder suggested that the ranking was best described as follows: RPL13 > EF-1α> HPRT > TUB > PP2A > PPIA > β2M > GAPDH > β-actin. Evaluations using geNorm, which is based on the idea that the lower the expression stability value (M), the better the stability of gene expression, suggested that the expression stability of these genes is best described as follows: RPL13 = EF-1α > β-actin > GAPDH > PP2A > TUB > PPIA > HPRT > β2M. Finally, RefFinder analysis showed that the comprehensive stability ranking of each gene was RPL13 > EF-1α > PP2A > HPRT > PPIA > TUB > β2M > β-actin > GAPDH. In addition, all of the paired coefficients of variation, Vn/(n+1), reference genes had little effect on the V value. The combination of the two best reference genes is a valid normalization strategy, and the results can be used to correct the expression levels of various target genes. Given this, we can conclude that RPL13 and EF-1α are the two best reference genes for adult tissues. These outcomes were then validated in a myod1 expression assay in the slow-twitch muscle, fast-twitch muscle, kidney, and gills. These experiments revealed no significant differences in relative outcomes when using RPL13, EF-1α, and their combination as reference genes, whereas significant differences were identified when using the three least stable genes β2M, β-actin and GAPDH. This result confirmed that stability evaluation using the four methods was both necessary and effective.
Based on our evaluations, we recommend that RPL13, EF-1α, and their combination are the ideal reference gene combinations for all previously evaluated adult tissue types in P. dentex, as verified by four individual algorithms. The results of this study provide the basis for improved standardization of qRT-PCR and transcriptomic evaluations in this species, which in turn should support more accurate evaluation of functional genes and provide technical support for the comprehensive and systematic molecular evaluation of more mature P. dentex samples. |
| Key words: Pseudocaranx dentex qRT-PCR Reference gene Stability |
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