Advances in Localization and Quantitation of Gene Transcripts by Nonradioactive In Situ Hybridization

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For a better understanding of the regulatory mechanism of celltype or tissue-type specific gene expression, it is essential to analyze the site and the amount of the expression of specific genes at a level of transcript by in situ hybridization (ISH). To get a precise localization of ISH signal at cellular and subcellular levels, a nonradioactive method using a hapten-labeled probe would be appropriate because of a high resolution. For a quantitative analysis of ISH signal, an amount of hapten residues in hybrids must be proportional to that of target mRNA. Considering both requirements, a hapten-labeled synthetic oligodeoxynucleotide (oligo-DNA) could be currently the best choice as probe nucleic acid. In fact, the use of digoxigenin (Dig) or thymine-thymine (T-T) dimer-labeled oligo-DNA, which was detected by HRP-anti-hapten, enabled us to analyze specific RNA signals at a subcellular level; estrogen receptor α mRNA was localized to the supranuclear region of glandular epithelial cells in monkey uterus and prolactin mRNA was predominantly found in the Golgi area of rat pituitary cells. Moreover, when we examined the localization of 28S rRNA and 18S rRNA in nucleoli of mouse Sertoli cells simultaneously using T-T dimerized 28S rRNA and Dig-labeled 18S rRNA oligo-DNA probes, followed by the detection with FITC-anti-(T-T dimer) and Rhodamine-anti-Dig, we found chimeric distribution of these rRNAs. Furthermore, pre-embedding electron-microscopic (EM) ISH revealed that 18S rRNA was distributed more widely than 28S rRNA in the nucleolus surrounding a fibrillar center. As a trial of quantitative analysis of specific transcripts by ISH, we performed dot-blot hybridization between c-myc sense oligo-DNA and T-T dimerized c-myc antisense oligo-DNA and measured the staining density of each dot by an image-analyzer, confirming the linearity in a range of less than 10^<10> c-myc DNA copies. Based upon the similar analysis of total RNA from 2.3×10^4 HL-60 cells, we calculated that the average number of c-myc mRNA per HL-60 cell was 439 copies. Finally, ISH was conducted on a cytospin preparation of HL-60 cells and the c-myc mRNA copy number of each HL-60 cell was successfully decided. Therefore, now we can analyze the colorimetric ISH signal quantitatively at an individual cell level by the use of an image-analyzer. On the other hand, in the case of EM-ISH, we tried to use colloidal gold particles as a quantitative signal; LR-white embedded sections of mouse testis were hybridized with rRNA oligo-DNA probes, and 28S rRNA and 18S rRNA signals were simultaneously detected with 10 nm and 15 nm gold particles, respectively. In accord with the results of the preembedding ISH, we found that 28S rRNA was abundant in the center part of the nucleolus, while 18S rRNA was dominant in the periphery, indicating that even though originated from the same precursor rRNA, the offsprings may have a different metabolic pathway. Collectively, the use of hapten-labeled synthetic oligo-DNA probe has tuned out to be appropriate for the quantitative analysis of ISH signals with a high enough resolution in both light- and electron-microscopic levels.
日本組織細胞化学会の論文

Advances in Localization and Quantitation of Gene Transcripts by Nonradioactive In Situ Hybridization

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