Diagnosis and Monitoring of PML-RAR-Positive Acute Promyelocytic Leukemia by Quantitative RT-PCR

This manuscript describes the application of QZyme™ reverse-transcription polymerase chain reaction (RT-PCR) to the quantification of PML-RARα transcripts as a marker of APL. QZyme™ is a method for real time detection and quantification of target genes or transcripts. The principle of QZyme analysis is similar to other quantitative PCR systems; however, the mechanism is quite different. QZyme exploits the catalytic activity of DNAzymes (deoxyribozymes), which are oligonucleotides that can bind and cleave nucleic acid substrates. The approach is well suited to monitoring minimal residual disease (MRD) in patients with APL, as a result of its ability to detect low numbers of transcripts and accurately measure differences in concentration over a broad dynamic range. Further, its capacity for duplex analysis has multiple advantages for analysis of clinical specimens.

Protocols for duplex, single-tube QZyme RT-PCR assays, which allow simultaneous quantification of PML-RARα fusion transcripts (either L-type and V-type, or S-type) and the internal control BCR transcript, are provided. These protocols can be used for analyzing patient RNA specimens and are suitable for clinical trial monitoring. For this type of work, it is recommended that investigators validate the assays to ensure reproducible, accurate, and specific results on the equipment in their own laboratories. Assay validation is critical for real-time quantitative RT-PCR (RQ-PCR) and is often overlooked. A guide to the steps involved in validation and recommendations for acceptance criteria is included in this chapter.