Co-detection of chimeric bcr/abl (target) and beta-actin (control) messenger RNA in individual CFU-GM colonies derived from CML patients using the polymerase chain reaction

Leuk Res. 1991;15(9):867-74. doi: 10.1016/0145-2126(91)90471-5.

Abstract

In order to quantitate the magnitude of the normal and Philadelphia (Ph') chromosome-positive(+) progenitor cells for various research and clinical settings/studies, we have applied the highly sensitive polymerase chain reaction (PCR) for examining the cells contained in individual hematopoietic colonies for chimeric bcr/abl mRNA, a specific molecular marker for chronic myelogenous leukemia (CML). Thus, individual 14-day CFU-GM colonies, obtained by growth of bone marrow cells from CML patients were removed from methylcellulose cultures and total RNA from each colony was isolated. First-strand complementary DNAs (cDNA) corresponding to all mRNAs in the sample were obtained by using random hexamers in a reverse transcription (RT) reaction. cDNA then served as the substrate in the PCR. To ensure the integrity of the RNA extracted from each colony, beta-actin and bcr/abl cDNA sequences were amplified in the same reaction vessel. Using this method, we have examined the colonies grown from three CML patients and found that 5 out of 5, 9 out of 9 and 8 out of 9 colonies contained a bcr/abl transcript. This method is simple, highly sensitive and should facilitate studies comparing the expression of various oncogenes in normal and leukemic hematopoietic progenitor cells.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Actins / biosynthesis
  • Actins / genetics
  • Biomarkers, Tumor
  • Blotting, Southern
  • Chimera
  • Electrophoresis, Agar Gel
  • Gene Rearrangement, B-Lymphocyte
  • Granulocytes / metabolism
  • Humans
  • In Vitro Techniques
  • Leukemia, Myelogenous, Chronic, BCR-ABL Positive / diagnosis
  • Leukemia, Myelogenous, Chronic, BCR-ABL Positive / genetics*
  • Leukemia, Myelogenous, Chronic, BCR-ABL Positive / metabolism
  • Macrophages / metabolism
  • Oligonucleotide Probes
  • Philadelphia Chromosome
  • Polymerase Chain Reaction
  • Protein-Tyrosine Kinases*
  • Proto-Oncogene Proteins / biosynthesis
  • Proto-Oncogene Proteins / genetics*
  • Proto-Oncogene Proteins c-abl / biosynthesis
  • Proto-Oncogene Proteins c-abl / genetics
  • Proto-Oncogene Proteins c-bcr
  • RNA, Messenger / analysis*
  • RNA, Messenger / genetics*

Substances

  • Actins
  • Biomarkers, Tumor
  • Oligonucleotide Probes
  • Proto-Oncogene Proteins
  • RNA, Messenger
  • Protein-Tyrosine Kinases
  • Proto-Oncogene Proteins c-abl
  • BCR protein, human
  • Proto-Oncogene Proteins c-bcr