Grillo M, Margolis F L
Department of Neurosciences, Roche Research Center, Roche Institute of Molecular Biology, Nutley, NJ 07110.
Biotechniques. 1990 Sep;9(3):262, 264, 266-8.
Our data demonstrate the use of the reverse transcriptase polymerase chain reaction (RT-PCR) technique to study mRNA expression of genes that are devoid of introns. We have developed conditions that eliminate the false positives that can result from any preexisting DNA and that could confuse the interpretation of results. This modification (DNase pretreatment under specified conditions) ensures that the product resulting from RT-PCR is due to amplification of cDNA that has been synthesized during the reverse transcriptase reaction. Our results illustrate and emphasize the importance of including both a DNase pretreatment and a minus RT control. Using this modified procedure, our data illustrate clearly the ability of this protocol to demonstrate the presence of very low levels of olfactory marker protein (OMP) mRNA in three non-olfactory rat brain regions (cerebellum, thalamus/hypothalamus and cerebral hemispheres) where OMP mRNA was previously unknown. These data confirm a prior report of the ectopic expression of OMP immunoreactivity in these locations and indicate for the first time the "illegitimate" expression of extremely low levels of OMP mRNA in a non-neural tissue. Finally, this modification of the RT-PCR procedure will now permit the study of expression of specific, rare, mRNA molecules in the absence of any prior knowledge of the structure of their genes of origin.
我们的数据展示了利用逆转录聚合酶链反应（RT-PCR）技术来研究无内含子基因的mRNA表达。我们已开发出一些条件，可消除因任何预先存在的DNA而可能导致的假阳性结果，这些假阳性结果可能会混淆结果的解读。这种改进（在特定条件下进行DNase预处理）确保了RT-PCR产生的产物是由于逆转录反应过程中合成的cDNA的扩增。我们的结果阐明并强调了同时包含DNase预处理和逆转录阴性对照的重要性。使用这种改进后的方法，我们的数据清楚地表明了该方案能够在三个非嗅觉大鼠脑区（小脑、丘脑/下丘脑和大脑半球）中检测到极低水平的嗅觉标记蛋白（OMP）mRNA的存在，而此前在这些区域OMP mRNA并不为人所知。这些数据证实了之前关于这些部位OMP免疫反应性异位表达的报道，并首次表明在非神经组织中存在极低水平的OMP mRNA的“异常”表达。最后，RT-PCR程序的这种改进现在将允许在对其起源基因结构毫无先验知识的情况下，研究特定的、罕见的mRNA分子的表达。
