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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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Hauptverfasser: Spiess, Birgit (VerfasserIn) , Kleiner, Helga (VerfasserIn) , Tarnopolscaia, Irina (VerfasserIn) , Naumann, Nicole (VerfasserIn) , Fabarius, Alice (VerfasserIn) , Hofmann, Wolf-Karsten (VerfasserIn) , Saußele, Susanne (VerfasserIn) , Seifarth, Wolfgang (VerfasserIn)
Dokumenttyp: Article (Journal)
Sprache:Englisch
Veröffentlicht: 2023
In: Cancers
Year: 2023, Jahrgang: 15, Heft: 15, Pages: 1-18
ISSN:2072-6694
DOI:10.3390/cancers15153914
Online-Zugang: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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Verfasserangaben:Birgit Spiess, Helga Kleiner, Irina Tarnopolscaia, Nicole Naumann, Alice Fabarius, Wolf-Karsten Hofmann, Susanne Saussele and Wolfgang Seifarth
Beschreibung
Zusammenfassung: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.
Beschreibung:Veröffentlicht: 1. August 2023
Gesehen am 25.09.2023
Beschreibung:Online Resource
ISSN:2072-6694
DOI:10.3390/cancers15153914

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