Normalizing complementary DNA by quantitative reverse transcriptase-polymerase chain reaction of beta(2)-microglobulin: Molecular monitoring of minimal residual disease in acute promyelocytic leukemia

Reverse transcription (RT)-polymerase chain reaction (PCR) raises unique me thodological matters that may hamper the reliability of the procedure, espe cially when results should direct therapeutic decisions. One of these matte rs is represented by the RT step. The present study shows that differences in complementary DNA (cDNA) preparations purposely containing increasing am ounts of retrotranscribed RNA were not disclosed by nonquantitative RT-PCR by two different housekeeping genes, leading to fictitious results when the expression or a given gene was quantitatively assessed. To overcome this p roblem, the following are proposed: 1) to evaluate the efficiency of RT ste p through the quantification, by competitive RT-PCR, of the expression leve ls of the housekeeping gene beta(2)-microglobulin (beta(2)M): 2) to normali ze each cDNA preparation to be comprised within I standard deviation of the mean value of beta(2)M absolute level (3.14 +/- 1.14 attomoles/mu g RNA) f ound by analyzing 33 cell lines of hematopoietic origin. To validate this s trategy in a clinical setting, serial cDNA samples from patients were check ed by conventional and quantitative RT-PCR for beta(2)M. Again, only a quan titative evaluation of beta(2)M levels was allowed to unveil significant di fferences, otherwise undetected, in the efficiency of RT reactions among th ese cDNA samples. Normalization of samples to obtain cDNA preparations cont aining comparable beta(2)M levels, eventually led to an increased sensitivi ty in the detection of PML-RAR alpha fusion transcripts. This approach seem s of great value for the monitoring of minimal residual disease in serial p atient samples when a tumor-specific marker is available.