Short, 12 mer fluorescently labeled methylphosphonated oligonucleotides to visualize beta-actin MRNA in vivo.
A pair of fluorescently labeled antisense ( complementary to beta-actin mRNA) or control methylphosphonated DNA 12-mers were introduced into live cells. After fixation their distribution throughout the cell was compared to the localization pattern for the pair of control oligos.The distribution of the two sets of oligos differed in that there was a distinct pool of antisense probes that were detected at elevated levels in the leading edge of fibroblast and cortical underlining. The resulting fluorescence patterns of antisense probes colocalized and were analogous to labeling pattern already described and produced by in situ hybridization. The length of each of the probe destabilized binding to mismatched sequences at physiological temperature, while the overall length of the pair gave a unique, highly sequence specific recognition of a target sequence. Simultaneous, in vivo application of multiple probes let include internal controls into the experimental setup, in order to distinguish different distributions of antisense and control probes in the same specimen.
Lazowski
K W
KW
Laboratory of Molecular Neurobiology, Nencki Institute, Warsaw, Poland. krzych@nencki.gov.pl
Kaczmarek
L
L
D016475:3T3 Cells; D000199:Actins / Q000235:genetics; D000818:Animals; D004247:DNA / Q000737:chemistry / Q000235:genetics; D005456:Fluorescent Dyes / Q000737:chemistry / Q000494:pharmacology; D017404:In Situ Hybridization, Fluorescence / Q000379:methods; D051379:Mice; D018715:Microscopy, Video / Q000379:methods; D009838:Oligodeoxyribonucleotides / Q000737:chemistry* / Q000235:genetics; D015345:Oligonucleotide Probes / Q000592:standards*; D010755:Organophosphates / Q000737:chemistry; D012333:RNA, Messenger / Q000032:analysis*
D000199:Actins; D005456:Fluorescent Dyes; D009838:Oligodeoxyribonucleotides; D015345:Oligonucleotide Probes; D010755:Organophosphates; D012333:RNA, Messenger; C027265:methylphosphate; D004247:DNA