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|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|
|Citation:||Clinical Chemistry, 2008; 54(9):1568-1571|
|Publisher:||Amer Assoc Clinical Chemistry|
|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|
|Appears in Collections:||Medicine publications|
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