抄録
Detection of specific mRNA signals using reverse transcription linked with polymerase chain reactions (RT/PCR) requires a minimum of two oligonucleotides used as sense and antisense primers for PCR. Identification of the resultant RT/PCR products is usually accomplished by either verifying digestion into the correct fragment sizes from a predicted restriction site internal to the amplified sequence or by hybridization using an oligonucleotide probe complementary to a portion of the amplified product.
In this report we describe an instance in which RT/ PCR of mRNA from cell lines (human fibroblasts and vascular smooth muscle), which did not express a given gene (von Willebrand factor), led to spurious amplification of cDNA products that appeared to have the correct predicted size on agarose gel electrophoresis and that also gave positive hybridization to probes made from PCR primers used for amplification. Cell lines (human endothelial cells) that did express the gene, however, gave positive hybridization signals for a probe complementary to a third region internal to the amplified sequence. This probe did not show any false hybridization to the amplified DNA products from fibroblasts and smooth muscle cells. This example indicates that three determinants (sense and antisense primers and predicted product size) are not enough to identify a true mRNA signal by RT/PCR. A fourth determinant, such as an oligonucleotide probe, needs to be employed to definitively identify RT/PCR amplification products.
