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 complementary DNA (GAPDH cDNA) as a template in ribonuclease (RNase) protection assays to measure canine GAPDH mRNA expression.


METHODS
Primers designed from the human GAPDH gene were used to amplify a 191-base pair canine GAPDH cDNA by reverse-transcription polymerase chain reaction. The cDNA was sequenced, and used as a template for RNase protection assay.


METHODS
Total RNA was isolated from a canine squamous carcinoma cell line.


RESULTS
Canine GAPDH cDNA had a high degree of homology to human, rat, and mouse GAPDH. In vitro transcription of canine GAPDH cDNA was used to produce complementary RNA that detected canine GAPDH mRNA by RNase protection assay.


CONCLUSIONS
Canine GAPDH cDNA is a useful loading control to be used in RNase protection assays measuring mRNA expression in canine cells or tissues.
Gröne
A
A
Department of Veterinary Biosciences, College of Veterinary Medicine, Ohio State University, Columbus 43210, USA.
Weckmann
M T
MT
Capen
C C
CC
Rosol
T J
TJ
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D004285:Dogs / Q000235:genetics*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000096:biosynthesis* / Q000235:genetics*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D012260:Ribonucleases; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic
D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D012260:Ribonucleases