Indirect RT-PCR in-situ hybridization: a novel non-radioactive method for detecting glucose-dependent insulinotropic peptide.
To establish indirect in-situ PCR for the detection of intestinal peptide hormones, rat intestine and a murine intestinal tumor cell line, STC 1, were used. The results exhibited intensive staining of GIP-producing K-cells. Paraformaldehyde-fixed cryostat sections yielded the best results in signal to background ratio with RT-PCR in-situ hybridization. Moreover, it was possible to elevate the positive staining signal and to reduce background staining. Digoxigenin-labeled in-situ hybridization served as a control for specificity and sensitivity of GIP (glucose-dependent insulinotropic peptide) mRNA expression on cryostat as well as paraffin sections. In conclusion, this RT-PCR in-situ hybridization protocol proves to be a specific, sensitive and reliable non-radioactive technique for the detection of intestinal peptide hormone mRNA, especially in tissues or tumor cells where the application of ISH is limited.
Steinhoff
M
M
Clinical Research Unit for Gastrointestinal Endocrinology, Department of Internal Medicine, Philipps-University Marburg, 35033 Marburg, Germany. msteinho@uni-muenster.de
Hesse
H
H
Göke
B
B
Steinhoff
A
A
Eissele
R
R
Slater
E P
EP
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D005749:Gastric Inhibitory Polypeptide / Q000032:analysis* / Q000235:genetics; D017403:In Situ Hybridization / Q000379:methods*; D007422:Intestines / Q000737:chemistry*; D051379:Mice; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction
D017931:DNA Primers; D005749:Gastric Inhibitory Polypeptide