Reverse Transcription Can Critically Impact the Diagnostic Outcome of BCR::ABL1 Quantitative Real-Time RT-PCR.

Reverse transcriptases (RT) are essential tools in fusion transcript monitoring in chronic myeloid leukemia (CML). The RT type and cDNA priming method may impair the stoichiometry of cDNA synthesis, thereby potentially introducing a bias in qRT-PCR data. Using the Acrometrix™ reference panel and 37 clinical specimens, we have comparatively investigated the performance of the RTs MLV and SuperScript IV with random hexamer vs. target-specific priming. Quantitative RT-PCR results identified the priming type and RT type as major factors for diagnostic data variation, mainly due to the different efficacies of processing low-copy-numbers (<50) compared to or high-copy targets. The impairment of SuperScript IV in processing low- and high-copy-number RNA targets equally was not reflected by the diagnostically relevant Log (%) values. Therefore, the correct representation of housekeeping and target genes should have priority when aiming at as high a number of housekeeping gene copies as possible. Our data suggest that for improving assay sensitivity, increased RNA/cDNA amounts and the use of distinct RT/priming combinations are advantageous. However, for inter-laboratory harmonization, the proper conversion factor according to the CML international standard (IS) has to be reevaluated each time the grade of RT is changed.