Detection of phosphatidylinositol glycan class A gene transcripts by RT in situ PCR hybridization. A comparative study using fluorescein, Texas Red, and digoxigenin-11 dUTP for color detection.
Gene-specific probes labeled with fluorescein, Texas Red, and digoxigenin-11 dUTP (DIG) were used for RT in situ PCR hybridization to detect PIG-A gene (phosphatidylinositol glycan class A) transcripts. The PIG-A gene is responsible for biosynthesis of the glycosylphosphatidyl-inositol (GPI) anchor. Lack of GPI anchor expression due to mutations can cause an acquired clonal hematologic disorder called paroxysmal nocturnal hemoglobinuria (PNH). In this RT in situ PCR study, two types of labeling methods, a direct method (using fluorescein and Texas Red) and an indirect method (using DIG-11 dUTP) were compared. Both were successfully applied to detect and localize the PIG-A gene transcripts within single cells of the cell lines AA2, H9, and JY. Furthermore, similar results for sensitivity and reproducibility were obtained. Advantages and disadvantages of the different labeling techniques are discussed. In addition, peripheral blood mononuclear cells from PNH patients were also included in this study.
Simon
S
S
Immuno A.G., Department of Oncogenic Viruses, Vienna, Austria.
Reipert
B
B
Eibl
M M
MM
Steinkasserer
A
A
D002478:Cells, Cultured; D003856:Deoxyuracil Nucleotides / Q000032:analysis; D004076:Digoxigenin / Q000031:analogs & derivatives / Q000032:analysis; D019793:Fluorescein / Q000032:analysis; D005456:Fluorescent Dyes / Q000032:analysis*; D015870:Gene Expression; D017261:Glycosylphosphatidylinositols / Q000235:genetics / Q000378:metabolism; D006457:Hemoglobinuria, Paroxysmal / Q000378:metabolism*; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods*; D007202:Indicators and Reagents / Q000032:analysis*; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D008214:Lymphocytes / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis*; D014158:Transcription, Genetic; D014966:Xanthenes / Q000032:analysis
D003856:Deoxyuracil Nucleotides; D005456:Fluorescent Dyes; D017261:Glycosylphosphatidylinositols; D007202:Indicators and Reagents; D008565:Membrane Proteins; D012333:RNA, Messenger; D014966:Xanthenes; C070202:digoxigenin-11-deoxyuridine triphosphate; C080086:phosphatidylinositol glycan-class A protein; C034657:Texas red; D004076:Digoxigenin; D019793:Fluorescein