Detection of cell specific cluster determinant expression by reverse transcriptase polymerase chain reaction.
We describe a new, rapid, sensitive, and reproducible method for examining gene expression of several cell specific surface cluster determinants, CD2, CD3-gamma, CD4, CD8-beta, CD14, CD19, CD20, CD23, and CD25-alpha, and terminal deoxynucleotidyl transferase which heretofore have been commonly detected by flow cytometry. The method presented uses the reverse transcriptase polymerase chain reaction (RT-PCR) to analyze CD gene expression in stable human cell lines, peripheral blood lymphocytes, bone marrow, and lymph node cells. Polymerase chain reaction products were quantitated by incorporation of radiolabeled nucleotide during PCR and the amount of nucleotide incorporated into DNA was measured by ion exchange filter chromatography. The usefulness of this methodology is demonstrated in an analysis of peripheral blood samples from a patient who presented with B cell deficiency. Results of analyses of peripheral blood samples from this patient by flow cytometry and RT-PCR are similar except that the increased sensitivity of RT-PCR permitted the detection of CD19, CD20, and CD23 in the blood samples of this patient who otherwise appeared to be lacking in all markers of B cell development.
James-Yarish
M
M
Department of Pediatrics, All Children's Hospital, University of South Florida, St. Petersburg 33701.
Bradley
W G
WG
Emmanuel
P J
PJ
Good
R A
RA
Day
N K
NK
D015703:Antigens, CD / Q000032:analysis* / Q000235:genetics; D001402:B-Lymphocytes / Q000276:immunology; D001483:Base Sequence; D002460:Cell Line; D004247:DNA / Q000032:analysis; D017931:DNA Primers; D005260:Female; D005434:Flow Cytometry; D015870:Gene Expression; D006801:Humans; D007153:Immunologic Deficiency Syndromes / Q000235:genetics / Q000276:immunology; D007223:Infant; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012194:RNA-Directed DNA Polymerase; D012680:Sensitivity and Specificity
D015703:Antigens, CD; D017931:DNA Primers; D004247:DNA; D012194:RNA-Directed DNA Polymerase