In situ detection of messenger RNA using digoxigenin-labeled oligonucleotides and rolling circle amplification.
The detection of specific RNA molecules in situ is routinely performed using haptenated probes, which are detected by either enzymatic amplification or direct fluorescence. A drawback of fluorescence labeling has been the reduced sensitivity relative to that of methods that use enzymes as signal generators. Reliable fluorescence detection methods often require the use of multiple oligonucleotide probes for each gene target. Here, we demonstrate that single haptenated DNA probes specific for actin mRNA may be detected in situ using antibody-coupled rolling circle amplification (immuno-RCA). This fluorescence-based detection method offers remarkable sensitivity due to the use of signal amplification and yet retains the ability to count hybridization signals as discrete objects. We demonstrate the detection of actin-specific immuno-RCA signals in the cytoplasm and use 3D image deconvolution of multiple z axis sections to show that there are hundreds of signals per cell. With some modifications, this method may be adaptable to the simultaneous detection of several RNA species, including low-copy-number mRNA.
Zhou
Y
Y
Department of Pathology, Yale University School of Medicine, 310 Cedar Street, New Haven, Connecticut, 06520, USA.
Calciano
M
M
Hamann
S
S
Leamon
J H
JH
Strugnell
T
T
Christian
M W
MW
Lizardi
P M
PM
D020121:5' Untranslated Regions / Q000235:genetics*; D000199:Actins / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D015342:DNA Probes; D004076:Digoxigenin; D007091:Image Processing, Computer-Assisted; D007074:Immunoglobulin G; D017403:In Situ Hybridization / Q000379:methods; D017404:In Situ Hybridization, Fluorescence; D008099:Liver / Q000166:cytology; D008853:Microscopy / Q000379:methods; D008969:Molecular Sequence Data; D020319:Oligodeoxyribonucleotides, Antisense; D015345:Oligonucleotide Probes; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D051381:Rats; D012680:Sensitivity and Specificity
D020121:5' Untranslated Regions; D000199:Actins; D015342:DNA Probes; D007074:Immunoglobulin G; D020319:Oligodeoxyribonucleotides, Antisense; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D004076:Digoxigenin