Comparison of 35S- and digoxigenin-labeled RNA and oligonucleotide probes for in situ hybridization. Expression of mRNA of the seminal vesicle secretion protein II and androgen receptor genes in the rat prostate.
The sensitivity of radiolabeled and digoxigenin-labeled RNA probes and synthetic oligonucleotide probes for the detection of seminal vesicle secretion protein II (SVS II) and androgen receptor (AR) mRNA was compared by in situ hybridization in paraformaldehyde-fixed cryostat sections of the rat prostate. Both genes are expressed in different amounts in the various prostatic lobes and contiguous glands. SVS II or AR RNA probes were either labeled with digoxigenin-11-UTP or [35S]UTP by in vitro transcription. A synthetic SVS II oligonucleotide probe was 3' end-labeled (tailed) with either digoxigenin-11-dUTP or [35S]dATP. Hybridized 35S-labeled probes were detected by autoradiography and digoxigenin-labeled probes by immunohistochemistry using alkaline phosphatase conjugated anti-digoxigenin antibody or gold-labeled antibody followed by protein A-gold and silver enhancement. Digoxigenin-labeled probes provided the same degree of sensitivity as their 35S-labeled counterparts for the detection by in situ hybridization of weakly and strongly expressed mRNA. Using both labeling methods, the SVS II RNA probes were more sensitive than the oligonucleotide probes and background labelling of the 35S-labeled oligonucleotide probe was high. The digoxigenin method produced less background with all probe types, hybridization signals showed higher resolution and results were obtained faster than with radiolabeled probes. The immunogold silver enhancement system provided the fastest detection of digoxigenin-labeled probes with a sensitivity and resolution similar to that provided by alkaline phosphatase anti-digoxigenin immunohistochemistry. It is concluded that digoxigenin probe labeling and detection provides a sensitive, reliable, and efficient alternative to radiolabeled probes for in situ hybridization of mRNA.
Komminoth
P
P
Department of Pathology, Tufts University School of Medicine, New England Medical Center Hospitals, Boston, MA 02111.
Merk
F B
FB
Leav
I
I
Wolfe
H J
HJ
Roth
J
J
D000818:Animals; D002369:Castration; D004076:Digoxigenin; D017403:In Situ Hybridization / Q000379:methods*; D008297:Male; D015345:Oligonucleotide Probes; D029584:Prostatic Secretory Proteins; D011506:Proteins / Q000032:analysis*; D015347:RNA Probes; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D011919:Rats, Inbred Strains; D011944:Receptors, Androgen / Q000032:analysis*; D029589:Seminal Plasma Proteins; D012669:Seminal Vesicles / Q000378:metabolism* / Q000648:ultrastructure; D013462:Sulfur Radioisotopes
D015345:Oligonucleotide Probes; D029584:Prostatic Secretory Proteins; D011506:Proteins; D015347:RNA Probes; D012333:RNA, Messenger; D011944:Receptors, Androgen; D029589:Seminal Plasma Proteins; D013462:Sulfur Radioisotopes; C043076:beta-microseminoprotein; D004076:Digoxigenin