Please use this identifier to cite or link to this item:
https://hdl.handle.net/2440/52531
Citations | ||
Scopus | Web of ScienceĀ® | Altmetric |
---|---|---|
?
|
?
|
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 1530-8561 |
Statement of Responsibility: | 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 |
Published version: | http://dx.doi.org/10.1373/clinchem.2008.105916 |
Appears in Collections: | Aurora harvest Medicine publications |
Files in This Item:
There are no files associated with this item.
Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.