Nordqvist K, Töhönen V
Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institutet, Stockholm, Sweden.
Int J Dev Biol. 1997 Aug;41(4):627-38.
The mRNA differential display technique has become a popular method for isolating novel genes in a variety of biological systems including carcinogenesis, hormone regulation, plant biology and neurobiology. We have further developed the method by optimizing different steps for the use of small amounts of material, such that differential display can be used in the study of developmental biology. Our techniques include a new assay for elimination of false positive cDNA clones and a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) method for the rapid analysis of differences in gene expression. This improved mRNA differential display strategy requires less than 4 microg of total RNA. We have used it for the isolation of genes which are expressed during gonad development in the mouse. One of the cDNAs found, cDNA 4.3 which corresponds to a part of the gene encoding the steroid hydroxylase 3betaHSD I, was shown to be a valuable marker for adrenal development and for Leydig cell differentiation and organization during testis development.
mRNA差异显示技术已成为在包括癌变、激素调节、植物生物学和神经生物学等多种生物系统中分离新基因的常用方法。我们通过优化使用少量材料的不同步骤进一步改进了该方法，从而使差异显示可用于发育生物学研究。我们的技术包括一种消除假阳性cDNA克隆的新检测方法和一种用于快速分析基因表达差异的半定量逆转录-聚合酶链反应（RT-PCR）方法。这种改进的mRNA差异显示策略所需的总RNA不到4微克。我们已将其用于分离在小鼠性腺发育过程中表达的基因。所发现的一个cDNA，即与编码类固醇羟化酶3βHSD I的基因的一部分相对应的cDNA 4.3，被证明是肾上腺发育以及睾丸发育过程中睾丸间质细胞分化和组织形成的一个有价值的标志物。
