Lin C, Spikings E, Zhang T, Rawson D
LIRANS Research Institute, University of Bedfordshire, 250 Butterfield, Great Marlings, Luton, Bedfordshire, LU2 8DL, UK.
Theriogenology. 2009 Apr 15;71(7):1147-55. doi: 10.1016/j.theriogenology.2008.12.013. Epub 2009 Feb 6.
Cryopreservation success is usually analysed in terms of cell survival, although there are other potential effects that do not necessarily result in cell death. These include DNA damage, which could result in altered gene expression. Real-time reverse transcriptase PCR allows quantitative analysis of gene expression but usually requires analysis of a 'housekeeping' gene as an internal reference. As the stability of housekeeping genes varies significantly among different groups of samples, it is recommended that those chosen are validated for each different type of sample group. This study aimed to validate housekeeping genes for use in cryopreservation studies of zebrafish embryos. Seven potential housekeeping genes were analysed across fresh and chilled intact embryos and across fresh and frozen isolated blastomeres using the GeNorm and NormFinder software packages. Results suggest that combined use of beta-actin and EF1alpha as housekeeping genes would be suitable for cryopreservation studies on zebrafish embryos and blastomeres.
冷冻保存的成功通常根据细胞存活率来分析，尽管还有其他一些潜在影响不一定会导致细胞死亡。这些影响包括DNA损伤，这可能导致基因表达改变。实时逆转录聚合酶链反应（Real-time reverse transcriptase PCR）可对基因表达进行定量分析，但通常需要分析一个“管家”基因作为内部参照。由于管家基因在不同样本组中的稳定性差异很大，建议针对每种不同类型的样本组对所选的管家基因进行验证。本研究旨在验证用于斑马鱼胚胎冷冻保存研究的管家基因。使用GeNorm和NormFinder软件包，对新鲜和冷藏的完整胚胎以及新鲜和冷冻的分离卵裂球中的七个潜在管家基因进行了分析。结果表明，联合使用β-肌动蛋白和EF1α作为管家基因适用于斑马鱼胚胎和卵裂球的冷冻保存研究。
