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.
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.
authors.affiliation: Division of Haematology, Institute of Medical & Veterinary Science, Adelaide, Australia. ross@imvs.sa.gov.au
authors.forename: David M
authors.initials: DM
authors.lastname: Ross
authors.forename: Dale B
authors.initials: DB
authors.lastname: Watkins
authors.forename: Timothy P
authors.initials: TP
authors.lastname: Hughes
authors.forename: Susan
authors.initials: S
authors.lastname: Branford
mesh_terms: D017931:DNA Primers / Q000032:analysis* / Q000235:genetics*; D016044:Fusion Proteins, bcr-abl / Q000032:analysis* / Q000235:genetics*; D006801:Humans; D015466:Leukemia, Myeloid, Chronic-Phase / Q000175:diagnosis / Q000235:genetics*; D018365:Neoplasm, Residual / Q000175:diagnosis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014158:Transcription, Genetic / Q000235:genetics*
chemical_list: D017931:DNA Primers; D016044:Fusion Proteins, bcr-abl