In situ hybridization in suspension and flow cytometry as a tool for the study of gene expression.
We describe a method to detect mRNA expression using in situ hybridization in suspension and flow cytometry. Our model was glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression in the leukemic cell line K562. A GAPDH cDNA probe was labeled with digoxigenin-11-dUTP and detected using an FITC-labeled anti-digoxigenin antiserum. We obtained good resolution in specific signals against background (GAPDH signal/control plasmid signal ratio +/- SE 3.5 +/- 0.9). The technique was optimized taking into account several hybridization variables, like fixation, hybridization time, effect of blocking agents, and stringency wash variations. This method also allowed us to quantitate the GAPDH RNA copy number/cell using a fluorescence standard; we obtained a figure of about 1200 copies/cell, which is in good agreement with the dot blot hybridization assay result. Flow cytometric analysis of in situ hybridization represents an original method to study gene expression. This technique has the potential to develop into a multiparametric tool for cell biology studies, examining specific mRNA production together with DNA content or membrane molecules expression, and offering the possibility to purify by sorting a cell population expressing a specific mRNA.
Lalli
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E
Laboratorio di Immunologia e Genetica, Istituti Ortopedici Rizzoli, Bologna, Italy.
Gibellini
D
D
Santi
S
S
Facchini
A
A
D001483:Base Sequence; D003587:Cytomegalovirus / Q000235:genetics; D015342:DNA Probes; D005434:Flow Cytometry / Q000379:methods; D016650:Fluorescein-5-isothiocyanate; D015870:Gene Expression; D005822:Genetic Vectors; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics*; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive; D008856:Microscopy, Fluorescence / Q000379:methods; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D016372:RNA, Antisense; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D015183:Restriction Mapping; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D015342:DNA Probes; D009838:Oligodeoxyribonucleotides; D016372:RNA, Antisense; D012333:RNA, Messenger; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D016650:Fluorescein-5-isothiocyanate