Use of reverse transcriptase polymerase chain reaction to monitor expression of intronless genes.
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.
Grillo
M
M
Department of Neurosciences, Roche Research Center, Roche Institute of Molecular Biology, Nutley, NJ 07110.
Margolis
F L
FL
D000818:Animals; D001709:Biotechnology; D001921:Brain / Q000378:metabolism; D015870:Gene Expression; D007438:Introns; D009419:Nerve Tissue Proteins / Q000235:genetics; D051842:Olfactory Marker Protein; D009831:Olfactory Mucosa / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase; D051381:Rats; D014018:Tissue Distribution
D009419:Nerve Tissue Proteins; D051842:Olfactory Marker Protein; C497599:Omp protein, rat; D012333:RNA, Messenger; D012194:RNA-Directed DNA Polymerase