CANINE GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE COMPLEMENTARY-DNA - POLYMERASE CHAIN-REACTION AMPLIFICATION, CLONING, PARTIAL SEQUENCE-ANALYSIS, AND USE AS LOADING CONTROL IN RIBONUCLEASE PROTECTION ASSAYS

Objective-To use canine glyceraldehyde-3-phosphate dehydrogenase compl ementary DNA (GAPDH cDNA) as a template in ribonuclease (RNase) protec tion assays to measure canine GAPDH mRNA expression. Design and Proced ure-Primers designed from the human GAPDH gene were used to amplify a 191-base pair canine GAPDH cDNA by reverse-transcription polymerase ch ain reaction. The cDNA was sequenced, and used as a template for RNase protection assay. Sample Population-Total RNA was isolated from a can ine squamous carcinoma cell line. Results-Canine GAPDH cDNA had a high degree of homology to human, rat, and mouse GAPDH. In vitro transcrip tion of canine GAPDH cDNA was used to produce complementary RNA that d etected canine GAPDH mRNA by RNase protection assay. Conclusion-Canine GAPDH cDNA is a useful loading control to be used in RNase protection assays measuring mRNA expression in canine cells or tissues.