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Type: Journal article
Title: Reverse transcription with random pentadecamer primers improves the detection limit of a quantitative PCR assay for BCR-ABL transcripts in chronic myeloid leukemia: Implications for defining sensitivity in minimal residual disease
Author: Ross, D.
Watkins, D.
Hughes, T.
Branford, S.
Citation: Clinical Chemistry (Washington, DC): international journal of molecular diagnostics and laboratory medicine, 2008; 54(9):1568-1571
Publisher: Amer Assoc Clinical Chemistry
Issue Date: 2008
ISSN: 0009-9147
Statement of
David M. Ross, Dale B. Watkins, Timothy P. Hughes and Susan Branford
Abstract: Background: Real-time quantitative reverse transcription PCR (RQ-PCR) assay for BCR-ABL is used to monitor treatment response in chronic myeloid leukemia (CML). BCR-ABL transcript levels decline over several years of imatinib treatment, and increasing numbers of patients have BCR-ABL transcripts at or below the limit of detection. More sensitive PCR methods are required to assess whether these patients have a long-term continuing decline in residual disease. Methods: We used random pentadecamer (R15) primers for reverse transcription in RQ-PCR and compared the results with our established method that uses random hexamers. An increase in assay sensitivity would be detected as an increase in the number of BCR-ABL transcripts. Results: BCR-ABL transcripts increased by 86% with R15 primers. We used R15 primers to retest 19 samples from selected CML patients who had no BCR-ABL transcripts recently detectable with hexamer primers and detected BCR-ABL transcripts in 68% of the samples. Use of R15 primers showed variable increases in the transcripts for control genes BCR (breakpoint cluster region), ABL1 (c-abl oncogene 1, receptor tyrosine kinase), and GUSB (glucuronidase, beta), depending on the gene examined. The reported BCR-ABL/control gene ratio was affected, and the estimated detection limit of the assay, which was based on increased control gene copy number, was different for each control gene. Conclusions: This simple modification to the reverse transcription methodology improved the detection limit of the RQ-PCR assay for BCR-ABL transcripts. In the field of CML, these results have important implications for defining the detection limit of an assay when the BCR-ABL transcript is undetectable. Random pentadecamer primers may also be useful in other reverse transcription PCR assays for which the abundance of the target RNA is low.
Keywords: Humans
Leukemia, Myeloid, Chronic-Phase
Neoplasm, Residual
Fusion Proteins, bcr-abl
DNA Primers
Sensitivity and Specificity
Reverse Transcriptase Polymerase Chain Reaction
Transcription, Genetic
DOI: 10.1373/clinchem.2008.105916
Appears in Collections:Aurora harvest
Medicine publications

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