Does this band make sense? Limits to expression based cancer studies.

Cancer researchers commonly employ reverse transcriptase polymerase chain reaction (RT-PCR) for gene expression analysis of cancer cells. While this technique is facile and reproducible, it is not without limitations. The human genome contains abundant nearly identical sequences (e.g. pseudogenes) to mRNA transcript sequences, which amplify when performing RT-PCR on samples with even trace amounts of genomic DNA. Such sequences include housekeeping transcripts such as beta-actin and GAPDH. This is also true for numerous gene products whose expression is altered in disease states such as cancer (e.g. pp32). Moreover, we describe that amplification of undesirable sequences is not simply avoided by designing primers spanning multiple exons. We also found that template-specific reverse transcriptase reactions lack the specificity necessary to definitively determine the sense or anti-sense orientation of an mRNA transcript. Given the above mentioned caveats and limitations of expression analysis studies, we encourage cancer investigators to test for the existence of intronless genomic sequences that are similar to the specific transcript of the gene being studied. Further, RNA samples should be completely genomic DNA-free prior to performing RT-PCR based assays. Finally, to ensure reliability of RT-PCR or array results, we recommend not utilizing the widely accepted loading controls, GAPDH and/or beta-actin.