Establishment of proliferative cell nuclear antigen gene as an internal reference gene for polymerase chain reaction of a wide range of archival and fresh mammalian tissues.
Polymerase chain reaction (PCR) from paraffin-embedded tissues provides a powerful tool to amplify DNA from a variety of recent and archival material. Because DNA from paraffin-embedded samples is more degraded than from fresh material, the amplification of reference genes is essential to exclude false-negative results. This study describes the use of the proliferative cell nuclear antigen (PCNA) gene as a reference gene in a range of animal species and in humans. The PCNA-PCR to amplify a fragment extending from exon 5 through exon 6 and including the intervening intron 6 gave a reproducible pattern, with a 280-base pair (bp) band from canine, equine, bovine, ovine, and caprine samples showing high sequence homology. Porcine, guinea pig, tiger, and lion samples, however, gave an additional fragment of approximately 197 bp. The whole intron 6 from these fragments is missing, possibly representing a pseudogene. In feline samples only the 197-bp fragment could be detected. This study shows that the PCNA gene is highly conserved across a broad range of animal species and is well suited as an internal control for PCR analysis in veterinary medicine.
Schiller
Irene
I
Institute of Veterinary Pathology, University of Zurich, Winterthurerstrasse 268, 8057 Zurich, Switzerland.
Lu
Zen Huat
ZH
Vaughan
Lloyd
L
Weilenmann
Roseline
R
Koundrioukoff
Stephane
S
Pospischil
Andreas
A
D000818:Animals; D001483:Base Sequence; D005188:False Negative Reactions; D008322:Mammals / Q000235:genetics*; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques; D010232:Paraffin; D016133:Polymerase Chain Reaction / Q000662:veterinary*; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics*; D012016:Reference Values; D015203:Reproducibility of Results; D013048:Specimen Handling; D016707:Tissue Fixation
D018809:Proliferating Cell Nuclear Antigen; D010232:Paraffin