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.
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.
Memorial Sloan-Kettering Cancer Center, New York, NY 10021.
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D
Wisniewski
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A
Strife
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B
Clarkson
D000199:Actins / Q000096:biosynthesis / Q000235:genetics; D014408:Biomarkers, Tumor; D015139:Blotting, Southern; D002678:Chimera; D004587:Electrophoresis, Agar Gel; D015322:Gene Rearrangement, B-Lymphocyte; D006098:Granulocytes / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000175:diagnosis / Q000235:genetics* / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism; D015345:Oligonucleotide Probes; D010677:Philadelphia Chromosome; D016133:Polymerase Chain Reaction; D011505:Protein-Tyrosine Kinases; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000235:genetics*; D016315:Proto-Oncogene Proteins c-abl / Q000096:biosynthesis / Q000235:genetics; D051562:Proto-Oncogene Proteins c-bcr; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics*
D000199:Actins; D014408:Biomarkers, Tumor; D015345:Oligonucleotide Probes; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D011505:Protein-Tyrosine Kinases; D016315:Proto-Oncogene Proteins c-abl; C496613:BCR protein, human; D051562:Proto-Oncogene Proteins c-bcr