Detection and localization of actin mRNA isoforms in chicken muscle cells by in situ hybridization using biotinated oligonucleotide probes.
We have developed in situ hybridization methodology for nonisotopically labeled oligonucleotide probes to detect cellular mRNA with improved speed, convenience, and resolution over previous techniques. Previous work using isotopically labeled oligonucleotide probes characterized important parameters for in situ hybridization (Anal Biochem 166:389, 1987). Eleven oligonucleotide probes were made to coding and noncoding regions of chick beta-actin mRNA and one oligonucleotide probe to chick alpha-cardiac actin mRNA. All the probes were 3' end-labeled with bio-11-dUTP using terminal transferase, and the labeled probes were hybridized to chicken myoblast and myotube cultures. The hybridized probe was detected using a streptavidin-alkaline phosphatase conjugate. Our assay for the success of probe hybridization and detection was the demonstration of beta-actin mRNA highly localized in the lamellipodia of single cells (Lawrence and Singer, Cell 45:407, 1986) as well as the expression of alpha-cardiac actin mRNA and the repression of beta-actin mRNA in differentiating myoblasts and in myotubes. With the alpha-cardiac probe, we found that this mRNA was distributed all over the cytoplasm of myotubes and differentiated (bipolar) single cells and negative in undifferentiated single cells and at the ends of myotubes. When beta-actin probes were used, two of 11 probes were highly sensitive, and, in pooling them together, the localization of beta-actin mRNA in fibroblastic single cells was evident at the leading edge of the motile cells, the lamellipodium. beta-Actin mRNA was not detected in myotubes except at the ends where contact was made with substrate. This indicates that both beta and cardiac actin mRNA can coexist in the same myotube cytoplasm but at different locations.
Taneja
K L
KL
Department of Cell Biology, University of Massachusetts Medical School, Worcester 01655.
Singer
R H
RH
D000199:Actins / Q000235:genetics*; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D001710:Biotin; D002478:Cells, Cultured; D002642:Chick Embryo; D003593:Cytoplasm / Q000737:chemistry; D007700:Kinetics; D009132:Muscles / Q000737:chemistry* / Q000166:cytology; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D011554:Pseudopodia / Q000737:chemistry; D012333:RNA, Messenger / Q000032:analysis*
D000199:Actins; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D001710:Biotin; D000469:Alkaline Phosphatase