Jonker A, de Boer P A, van den Hoff M J, Lamers W H, Moorman A F
Department of Anatomy and Embryology, Academical Medical Centre, University of Amsterdam, The Netherlands.
J Histochem Cytochem. 1997 Mar;45(3):413-23. doi: 10.1177/002215549704500309.
In situ hybridization analysis of tissue mRNA concentrations remains to be accepted as a quantitative technique, even though exposure of tissue sections to photographic emulsion is equivalent to Northern blot analysis. Because of the biological importance of in situ quantification of RNA sequences within a morphological context, we evaluated the quantitative aspects of this technique. In calibrated microscopic samples, autoradiographic signal (density of silver grains) was proportionate to the radioactivity present, to the exposure time, and to time of development of the photographic emulsion. Similar results were obtained with tissue sections, showing that all steps of the in situ hybridization protocol, before and including the detection of the signal, can be reproducibly performed. Furthermore, the integrated density of silver grains produced in liver and intestinal sections by the in situ hybridization procedure using 35S-labeled riboprobes is directly proportionate to the signal obtained by quantitative Northern blot analysis. The significance of this finding is that in situ quantification of RNA can be realized with high sensitivity and with the additional advantage of the possibility of localizing mRNA within the cells of interest. Application of this procedure on fetal and adult intestinal tissue showed that the carbamoylphosphate synthetase (CPS)-expressing epithelial cells of both tissues accumulated CPS mRNA to the same level but that whole-organ CPS mRNA levels decreased four-to fivefold in the same period, owing to a comparable decrease in the number of CPS-expressing cells in total intestinal tissue.
尽管将组织切片与感光乳剂曝光等同于Northern印迹分析，但组织mRNA浓度的原位杂交分析仍未被视为一种定量技术。由于在形态学背景下对RNA序列进行原位定量具有生物学重要性，我们评估了该技术的定量方面。在校准的显微镜样本中，放射自显影信号（银颗粒密度）与存在的放射性、曝光时间以及感光乳剂的显影时间成正比。在组织切片中也获得了类似的结果，表明原位杂交方案中信号检测之前及包括信号检测在内的所有步骤都可以重复进行。此外，使用35S标记的核糖探针通过原位杂交程序在肝脏和肠道切片中产生的银颗粒积分密度与定量Northern印迹分析获得的信号直接成正比。这一发现的意义在于，可以高灵敏度地实现RNA的原位定量，并且还有一个额外的优势，即有可能在感兴趣的细胞内定位mRNA。将该程序应用于胎儿和成人肠道组织表明，两种组织中表达氨甲酰磷酸合成酶（CPS）的上皮细胞积累CPS mRNA的水平相同，但由于整个肠道组织中表达CPS的细胞数量有相当程度的减少，同期全器官CPS mRNA水平下降了四至五倍。
