Reverse transcription can critically impact the diagnostic outcome of BCR::ABL1 quantitative real-time RT-PCR
Reverse transcriptases (RT) are essential tools in BCR::ABL1 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 BCR::ABL1 qRT-PCR data. Using the Acrometrix™ BCR...
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| Main Authors: | , , , , , , , |
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| Format: | Article (Journal) |
| Language: | English |
| Published: |
2023
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| In: |
Cancers
Year: 2023, Volume: 15, Issue: 15, Pages: 1-18 |
| ISSN: | 2072-6694 |
| DOI: | 10.3390/cancers15153914 |
| Online Access: | Verlag, kostenfrei, Volltext: https://doi.org/10.3390/cancers15153914 Verlag, kostenfrei, Volltext: https://www.mdpi.com/2072-6694/15/15/3914 
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| Author Notes: | Birgit Spiess, Helga Kleiner, Irina Tarnopolscaia, Nicole Naumann, Alice Fabarius, Wolf-Karsten Hofmann, Susanne Saussele and Wolfgang Seifarth |
| Summary: | Reverse transcriptases (RT) are essential tools in BCR::ABL1 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 BCR::ABL1 qRT-PCR data. Using the Acrometrix™ BCR::ABL1 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 BCR::ABL1 low-copy-numbers (<50) compared to GUSB or ABL1 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 (BCR::ABL1/GUSB%) values. Therefore, the correct representation of housekeeping and BCR::ABL1 target genes should have priority when aiming at as high a number of housekeeping gene copies as possible. Our data suggest that for improving BCR::ABL1 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. |
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| Item Description: | Veröffentlicht: 1. August 2023 Gesehen am 25.09.2023 |
| Physical Description: | Online Resource |
| ISSN: | 2072-6694 |
| DOI: | 10.3390/cancers15153914 |