Indirect RT-PCR in-situ hybridization: a novel non-radioactive method for detecting glucose-dependent insulinotropic peptide

Regul Pept. 2001 Mar 2;97(2-3):187-94. doi: 10.1016/s0167-0115(00)00219-6.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • DNA Primers
  • Gastric Inhibitory Polypeptide / analysis*
  • Gastric Inhibitory Polypeptide / genetics
  • In Situ Hybridization / methods*
  • Intestines / chemistry*
  • Mice
  • Rats
  • Reverse Transcriptase Polymerase Chain Reaction*

Substances

  • DNA Primers
  • Gastric Inhibitory Polypeptide