As with all nucleic acid-based tests, a variety of controls are required to ensure that reliable results are produced. A positive control must be included, and may be diluted to allow assessment of the assay sensitivity for each run. Negative controls also are critical and should provide controls for amplicon carryover during sample preparation and amplification. Perhaps the most critical control for RT-PCR assays is an internal control to demonstrate the presence of amplifiable mRNA in each sample. The lability of mRNA, and the challenge of procuring clinical samples for processing in a timely fashion, makes this an important measure of the suitability of samples for analysis.
Target choice is critical and must be able to distinguish between the tumor cells and other cells in the sample. Assessment of assay specificity is a critical task in development of an RT-PCR assay, and must include analysis of a large number of clinical samples taken from patients without disease as well as with benign disease of the organ from which the tumor has developed. Numerous commonly used mRNA targets are known to potentially lack specificity, such as cytokeratin 19, which is reported to be expressed in normal leukocytes.21 As has been mentioned, immunoselection of the tumor cells is one approach to avoid false-positive results in this setting. Pseudogenes for cytokeratin 19 and other genes can give rise to amplicons that appear by size to be derived from mRNA, but in fact reflect contaminating DNA present in the processed sample. Amplification reactions must be designed to detect gene transcripts rather than the gene itself, by using PCR primers that span intron/exon boundaries.
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