A validation of 10 feline reference genes for gene expression measurements in snap-frozen tissues.
For a proper determination of relative mRNA expression levels with real-time quantitative PCR (Q-PCR) internal standards, such as the expression of reference genes, are of utmost importance. For cats, in contrast to dogs, no validation of reference genes has been published. Our goal was to evaluate frequently used reference genes for the analysis of relative mRNA levels from feline tissues in a SYBR Green-based Q-PCR protocol. First, primers were optimized on mRNA-derived cDNA from liver and kidney tissues of randomly chosen (healthy and diseased) cats. Then, the expression variation and stability of each reference gene within a specific tissue was determined. Dental roots and crowns, heart (left ventricle), renal, liver, lung, and mammary gland tissues from 3 to 11 cats of different breeds, sexes, ages, and disease status were included in this study. Averaging relative stabilities over these six tissues revealed the usefulness of each tested gene as reference gene. In order to compensate for the expression variation of a reference gene within a specific tissue, as much as six reference genes (e.g. RPL17, RPL30, RPS7, YWHAZ, and HPRT) were required to obtain highly reliable data in cat tissues. The optimal set of reference genes depended on the tissue analyzed and should, ideally, be selected and evaluated at the start of each experimental condition. A comparison with a similar evaluation in dogs revealed three issues: (i) most ribosomal genes are suitable in both species; (ii) good non-ribosomal reference genes differ; (iii) more feline than canine reference genes are required for proper analysis.
Penning
Louis C
LC
Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 104, PO Box 80154, 3508 TD Utrechet, The Netherlands. L.C.Penning@uu.nl <L.C.Penning@uu.nl>
Vrieling
Henriette E
HE
Brinkhof
Bas
B
Riemers
Frank M
FM
Rothuizen
Jan
J
Rutteman
Gerard R
GR
Hazewinkel
Herman A W
HA
D000818:Animals; D002415:Cats / Q000235:genetics*; D015925:Cryopreservation; D020869:Gene Expression Profiling / Q000379:methods* / Q000662:veterinary*; D007668:Kidney; D008099:Liver; D008168:Lung; D008321:Mammary Glands, Animal; D009206:Myocardium; D020411:Oligonucleotide Array Sequence Analysis; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D012016:Reference Values; D015203:Reproducibility of Results; D014070:Tooth
D012333:RNA, Messenger