Nonradioactive, sequence-specific detection of RNA in situ by primed in situ labeling (PRINS).
A new method for the detection of RNA in situ is presented. It is based on sequence-dependent annealing of unlabeled specific oligonucleotide primers to intracellular RNA and subsequent chain elongation catalyzed by reverse transcriptase. Under the conditions described, biotin-labeled nucleotides can be incorporated and the cDNA synthesized in situ can thus be detected using fluorescein-conjugated avidin. Compared to traditional in situ hybridization the use of short oligonucleotide primers has the potential advantage of being better to discriminate between closely related RNA transcripts. Compared to in situ transcription with radioactive precursors we find it more attractive to use fluorescein-conjugated avidin as detection system because it allows a more detailed study of cell and signal simultaneously.
Mogensen
J
J
Institute for Human Genetics, University of Aarhus, Denmark.
Kølvraa
S
S
Hindkjaer
J
J
Petersen
S
S
Koch
J
J
Nygård
M
M
Jensen
T
T
Gregersen
N
N
Junker
S
S
Bolund
L
L
D000818:Animals; D001345:Autoradiography; D001360:Avidin; D001483:Base Sequence; D004273:DNA, Neoplasm / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D019793:Fluorescein; D005452:Fluoresceins; D008856:Microscopy, Fluorescence / Q000379:methods; D008969:Molecular Sequence Data; D009101:Multiple Myeloma / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D009841:Oligonucleotides / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D012334:RNA, Neoplasm / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D004273:DNA, Neoplasm; D005452:Fluoresceins; D009841:Oligonucleotides; D012334:RNA, Neoplasm; D001360:Avidin; D019793:Fluorescein