Bhat Irfan Ahmad, Dubiel Milena Malgorzata, Rodriguez Eduardo, Jónsson Zophonías Oddur
Institute of Life and Environmental Sciences, School of Engineering and Natural Sciences, University of Iceland, 101 Reykjavik, Iceland.
Benchmark Genetics, 220 Hafnarfjordur, Iceland.
Animals (Basel). 2023 Mar 19;13(6):1094. doi: 10.3390/ani13061094.
The challenge in extracting high-quality RNA impedes the investigation of the transcriptome of developing salmonid embryos. Furthermore, the mRNA expression pattern of important PGC and SD genes during the initial embryonic development of is yet to be studied. So, in the present study, we aimed to isolate high-quality RNA from eggs and developing embryos to check , , , , , , , and expression by qPCR. Additionally, four HKGs (, , and were validated to select the best internal control for qPCR. High-quality RNA was extracted, which was confirmed by spectrophotometer, agarose gel electrophoresis and Agilent TapeStation analysis. was chosen as a reference gene because it exhibited lower intra- and inter-sample variation. transcripts were expressed in all the developmental stages, while was expressed only up to 40 d°C. was expressed in later stages and remained at its peak for a shorter period, while showed an irregular pattern of mRNA expression. The mRNA expression levels of SD genes were observed to be upregulated during the later stages of development, prior to hatching. This study presents a straightforward methodology for isolating high-quality RNA from salmon eggs, and the resulting transcript profiles of significant PGC and SD genes in could aid in improving our comprehension of reproductive development in this commercially important species.
提取高质量RNA的挑战阻碍了对鲑科胚胎发育转录组的研究。此外,重要的原始生殖细胞(PGC)和性别决定(SD)基因在鲑科胚胎初始发育过程中的mRNA表达模式尚未得到研究。因此,在本研究中,我们旨在从卵和发育中的胚胎中分离高质量RNA,通过实时定量PCR(qPCR)检测[多个基因名称未给出]的表达。此外,对四个内参基因([多个基因名称未给出])进行了验证,以选择用于qPCR的最佳内参。提取了高质量RNA,通过分光光度计、琼脂糖凝胶电泳和安捷伦 TapeStation 分析进行了确认。[某个基因名称未给出]被选为参考基因,因为它在样本内和样本间表现出较低的变异性。[某个基因名称未给出]转录本在所有发育阶段均有表达,而[某个基因名称未给出]仅在40日龄时表达。[某个基因名称未给出]在后期阶段表达,并在较短时间内达到峰值,而[某个基因名称未给出]表现出不规则的mRNA表达模式。观察到SD基因的mRNA表达水平在发育后期、孵化前上调。本研究提出了一种从鲑鱼卵中分离高质量RNA的简单方法,所得的鲑科重要PGC和SD基因的转录谱有助于提高我们对这个商业上重要物种生殖发育的理解。