Sellars Melony J, Vuocolo Tony, Leeton Lisa A, Coman Greg J, Degnan Bernard M, Preston Nigel P
CSIRO Food Futures National Research Flagship, Australia; CSIRO Marine and Atmospheric Research, 233 Middle Street, Cleveland, Qld 4163, Australia.
J Biotechnol. 2007 May 1;129(3):391-9. doi: 10.1016/j.jbiotec.2007.01.029. Epub 2007 Feb 9.
Housekeeping genes are often used as references when quantifying the relative abundance of transcripts of interest, because it is assumed that they are stably expressed across tissues and developmental stages. Standard housekeeping genes are targeted particularly in organisms where there is no detailed information on gene expression profiles. Here, the validity of using the two widely accepted housekeeping genes, 18S rRNA and beta-actin, as reference genes to normalize real-time RT-PCR gene expression data from the Kuruma shrimp, Marsupenaeus japonicus, was tested. Expression patterns of two target genes in a diverse sample set of embryonic, larval, post-larval and gonad mRNAs were quantified using relative and absolute real-time RT-PCR procedures. Comparison of these approaches revealed significant differences (P<0.0001) in transcript level profiles between the relative and absolute procedures for both target genes. When 18S rRNA was used as a reference, target gene expression was more similar to that of the absolute method than when beta-actin was used as a reference. Variability between the relative and absolute procedures occurred for a greater percentage of the embryonic stages compared to later developmental stages. This study indicates that the use of 18S rRNA and beta-actin for studying relative gene expression patterns in Kuruma shrimp embryonic, larval, post-larval and gonad samples will give significantly variable results, and illustrates the proposition that housekeeping genes are not necessarily appropriate references for real-time RT-PCR data normalization. Until suitable reference genes are characterized, gene expression experiments using the studied Kuruma shrimp tissues of different morphological developmental stages should use absolute quantification procedures.
在定量分析感兴趣的转录本相对丰度时，管家基因常被用作参考，因为假定它们在不同组织和发育阶段均稳定表达。标准管家基因尤其适用于那些没有详细基因表达谱信息的生物体。在此，我们测试了使用两种广泛认可的管家基因18S rRNA和β-肌动蛋白作为参考基因，对日本囊对虾（Marsupenaeus japonicus）实时逆转录聚合酶链反应（RT-PCR）基因表达数据进行标准化的有效性。使用相对和绝对实时RT-PCR程序，对一组包含胚胎、幼虫、幼体后期和性腺mRNA的多样样本中两个靶基因的表达模式进行了定量分析。这些方法的比较显示，两个靶基因的相对和绝对程序之间，转录本水平谱存在显著差异（P<0.0001）。当使用18S rRNA作为参考时，靶基因表达比使用β-肌动蛋白作为参考时更类似于绝对方法。与后期发育阶段相比，相对和绝对程序之间的变异性在胚胎阶段所占百分比更大。本研究表明，在研究日本囊对虾胚胎、幼虫、幼体后期和性腺样本中的相对基因表达模式时，使用18S rRNA和β-肌动蛋白会得出显著不同的结果，并说明了管家基因不一定适合用于实时RT-PCR数据标准化这一观点。在鉴定出合适的参考基因之前，对处于不同形态发育阶段的日本囊对虾组织进行基因表达实验时，应使用绝对定量程序。
