Guiot Y, Rahier J
Department of Pathology, School of Medicine, University of Louvain, Belgium.
Diagn Mol Pathol. 1997 Oct;6(5):261-6. doi: 10.1097/00019606-199710000-00003.
The recent improvement of in situ hybridization (ISH) procedures, the increased sensitivity of immunohistochemical detection systems, and the development of assisted image analysis now enable the quantification of specific messenger RNAs (mRNAs) detected by nonisotopic probes on histological sections. However, the reliability and accuracy of this type of mRNA quantification are still to be determined. To this end, we compared in an experimental model of rat malnutrition the densitometric analysis of proinsulin mRNA detected by nonradioactive ISH with those obtained from radioactive Northern blot hybridization (NBH). Proinsulin gene expression was quantified by ISH and by NBH in the pancreatic islets of normally fed rats, rats fasted for 3 days, and rats refed for 8, 24, and 48 h after fasting. Starvation decreased the pancreatic proinsulin mRNA signal by 34% and 38%, as evaluated by ISH and NBH, respectively. Also, with both methods, mRNA levels returned to normal after refeeding. Taken together, the results derived from nonradioactive quantitative ISH were closely correlated to those obtained by quantitative NBH (r = 0.975, p < 0.005). It is thus possible to evaluate variations of mRNA content accurately by quantitative ISH as is currently done by NBH, but with the invaluable advantage of integrating the data with a morphological analysis.
近期原位杂交（ISH）技术的改进、免疫组织化学检测系统灵敏度的提高以及辅助图像分析技术的发展，使得在组织学切片上能够对非同位素探针检测到的特定信使核糖核酸（mRNA）进行定量分析。然而，这种mRNA定量分析的可靠性和准确性仍有待确定。为此，我们在大鼠营养不良的实验模型中，将非放射性ISH检测到的胰岛素原mRNA的光密度分析结果与放射性Northern印迹杂交（NBH）得到的结果进行了比较。在正常喂养的大鼠、禁食3天的大鼠以及禁食后再喂养8、24和48小时的大鼠的胰岛中，通过ISH和NBH对胰岛素原基因表达进行了定量分析。饥饿使胰腺胰岛素原mRNA信号分别通过ISH和NBH评估降低了34%和38%。同样，两种方法均显示，再喂养后mRNA水平恢复正常。总体而言，非放射性定量ISH得到的结果与定量NBH得到的结果密切相关（r = 0.975，p < 0.005）。因此，如同目前通过NBH所做的那样，通过定量ISH能够准确评估mRNA含量的变化，但具有将数据与形态学分析相结合这一宝贵优势。
