Gromova I, Gromov P, Celis J E
Department of Medical Biochemistry and Danish Centre for Human Genome Research, The University of Aarhus, Aarhus C.
Electrophoresis. 1999 Feb;20(2):241-8. doi: 10.1002/(SICI)1522-2683(19990201)20:2<241::AID-ELPS241>3.0.CO;2-A.
Differential display in combination with arbitrarily primed polymerase chain reaction (PCR) fingerprinting has become one of the most powerful techniques to identify and isolate mRNAs that are differentially expressed in pairs of biological samples. However, in many cases the cDNA band corresponding to the differentially amplified product contains several cDNA species that comigrate with the cDNA of interest due to the poor resolution of the fingerprinting gels, thus hampering further analysis and identification of the desirable cDNA. To improve the electrophoretic resolution of differentially amplified cDNAs, we have utilized Resolver Gold agarose gel electrophoresis (Ingenius) as an additional step to overcome downstream problems encountered during RNA fingerprinting experiments. To illustrate the power of the modified differential display procedure we present a detailed analysis of the cDNA products differentially displayed in tumor biopsies obtained from a noninvasive (grade II, Ta) and an invasive (grade III, T2-T4) human bladder transitional cell carcinoma (TCC). Several genes that were differentially expressed in this tumor pair were identified. These included: tropomyosin 4, the protein disulfide isomerase precursor (PDI), MRP14, signal transducer CD24, keratins 8 and 13, cytochrome oxidase subunit IV (COXIV), putative transcription factor HOX-1.3, as well as two novel genes of yet unknown function. All of the identified cDNAs were shown to be truly differentially expressed by Northern blotting, reverse transcriptase-PCR (RT-PCR), and two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) analysis of the corresponding lesions.
差异显示结合任意引物聚合酶链反应（PCR）指纹图谱技术，已成为鉴定和分离在成对生物样品中差异表达的mRNA的最强大技术之一。然而，在许多情况下，由于指纹图谱凝胶分辨率较差，与差异扩增产物对应的cDNA条带包含几种与目标cDNA一起迁移的cDNA种类，从而妨碍了对所需cDNA的进一步分析和鉴定。为了提高差异扩增cDNA的电泳分辨率，我们使用了Resolver Gold琼脂糖凝胶电泳（Ingenius）作为额外步骤，以克服RNA指纹图谱实验中遇到的下游问题。为了说明改进后的差异显示方法的强大功能，我们对从非侵袭性（II级，Ta）和侵袭性（III级，T2 - T4）人类膀胱移行细胞癌（TCC）获得的肿瘤活检组织中差异显示的cDNA产物进行了详细分析。鉴定出了在这对肿瘤中差异表达的几个基因。这些基因包括：原肌球蛋白4、蛋白质二硫键异构酶前体（PDI）、MRP14、信号转导子CD24、角蛋白8和13、细胞色素氧化酶亚基IV（COXIV）、假定转录因子HOX - 1.3，以及两个功能未知的新基因。通过Northern印迹、逆转录酶PCR（RT - PCR）以及对相应病变的二维聚丙烯酰胺凝胶电泳（2 - D PAGE）分析，所有鉴定出的cDNA均显示为真正的差异表达。
