Sensitive detection of rat gastrin mRNA by in situ hybridization with chemically biotinylated oligodeoxynucleotides: validation, quantitation, and double-staining studies.
Chemically biotin-labeled oligonucleotides form attractive reagents, as large quantities of stable and well-defined probes can easily be produced. Their usefulness for in situ hybridization was tested using rat gastrin cells as a model. Two probes recognizing two different regions of rat gastrin mRNA were synthesized and produced specific and equally strong hybridization signals. A probe complementary to human gastrin mRNA, but with mismatches to the rat gastrin mRNA sequence, failed to reveal rat gastrin cells under the stringency conditions used. Northern blotting revealed that the rat gastrin mRNA probes reacted exclusively with the appropriately sized (approximately 650 bases) mRNA. Model systems demonstrated that the hybridization signal, as revealed by alkaline phosphatase-based detection, varied linearly with the 10logarithm of target concentration and also showed that a new detection system was much more sensitive than previously used systems. In agreement with previous biochemical data, image analysis showed that starvation of rats led to a progressive decrease in cell staining intensities and cell numbers. Double staining for rat gastrin mRNA and gastrin immunoreactivity showed that in adult rats almost all gastrin cells expressed both mRNA and protein. Similar studies on developing rat gastrin cells revealed discrepancies between gastrin mRNA and gastrin-immunoreactive cells during the first week of newborn life. Subsequently, expression of mRNA and protein in the cells became gradually more concordant.
Larsson
L I
LI
Department of Molecular Cell Biology, State Serum Institute, Copenhagen, Denmark.
Hougaard
D M
DM
D000818:Animals; D000831:Animals, Newborn; D001483:Base Sequence; D005753:Gastric Mucosa / Q000737:chemistry* / Q000166:cytology / Q000254:growth & development; D005755:Gastrins / Q000032:analysis* / Q000235:genetics; D007124:Immunoenzyme Techniques; D017403:In Situ Hybridization; D008297:Male; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D012333:RNA, Messenger / Q000032:analysis*; D051381:Rats; D017208:Rats, Wistar; D012680:Sensitivity and Specificity; D013217:Starvation
D005755:Gastrins; D015345:Oligonucleotide Probes; D012333:RNA, Messenger