RTPCR Screening

An important problem with RT-PCR is the potential for false-positive results due to PCR contamination. To avoid this, stringent controls must be performed and ideally RNA extraction should be performed in a laboratory remote from that in which PCR is performed. Moreover, preparation of RT and PCR master mixes must be physically separated from areas in which PCR products are handled. Less well appreciated is the potential for molecular screening using RT-PCR to yield false-negative results in the presence of rare or atypical breakpoints. For example, molecular screening strategies for CBFB-MYH11 typically involve detection of type A, D, and E breakpoints, which together account for approximately 90% of cases; hence, it is possible that in rare instances, cases with alternative breakpoints that lack inv(16)/t(16;16) could be missed. Similarly, infrequent cases of PML-RARA-associated APL with atypical breakpoints are not detected by some standard RT-PCR

primer sets; however, such cases are unlikely to be missed, given the typical morphological features, and will give a positive result (microparticulate pattern) in the PML immunofluorescence test. False-negative results are also a potential problem in the presence of poor-quality RNA or inefficiency of the RT step.

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