Schmidt B F, Chao J, Zhu Z, DeBiasio R L, Fisher G
Center for Light Microscope, Imaging and Biotechnology, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, USA.
J Histochem Cytochem. 1997 Mar;45(3):365-73. doi: 10.1177/002215549704500304.
We demonstrate that the CAtalyzed Reporter Deposition method (CARD), utilizing the novel fluorescent reporter Cy3.29-tyramide, is successful in the Fluorescent in Situ Hybridization (FISH) detection of RNA and single-copy DNA. Histone 4 expression is detected in RNA extracts of 5-phase, synchronized HeLa cells by dot-blot analysis. Gene expression of histone 4 in HeLa cells is demonstrated by FISH via CARD, utilizing oligonucleotide probes. Fluorescence intensity measurements on CARD-amplified histone 4 RNA detection showed (a) a 25-fold amplification of the signal brightness by biotinylated oligonucleotide probes and (b) a sixfold amplification of the signal brightness by horseradish peroxidase (HRP)-labeled histone 4 probes vs the directly stained control. The sensitivity of the CARD method is demonstrated by the FISH detection of single-copy DNA on human corneal fibroblast and HeLa S5 interphase nuclei. Chromosomal localization of the single copy DNA is demonstrated on HeLa S3 metaphase chromosome spreads.
我们证明，利用新型荧光报告分子Cy3.29-酪胺的催化报告分子沉积法（CARD）在RNA和单拷贝DNA的荧光原位杂交（FISH）检测中是成功的。通过斑点印迹分析在5期同步化的HeLa细胞的RNA提取物中检测到组蛋白4的表达。利用寡核苷酸探针，通过CARD在FISH中证明了HeLa细胞中组蛋白4的基因表达。对CARD扩增的组蛋白4 RNA检测的荧光强度测量显示：（a）生物素化寡核苷酸探针使信号亮度放大25倍；（b）与直接染色对照相比，辣根过氧化物酶（HRP）标记的组蛋白4探针使信号亮度放大6倍。通过对人角膜成纤维细胞和HeLa S5间期细胞核上单拷贝DNA的FISH检测证明了CARD方法的灵敏度。在HeLa S3中期染色体铺片上证明了单拷贝DNA的染色体定位。
