Technical note: validation of internal control genes for gene expression analysis in bovine polymorphonuclear leukocytes.
Analysis of gene expression is becoming more important in all areas of biological research to evaluate gene expression during physiological and pathological conditions (e.g., mastitis), not the least in the field of animal research. Presently, real-time gene expression analysis is considered to be the method of choice for accurate and sensitive quantification of mRNA transcripts. Because comparison of gene expression levels is frequently the aim of these experiments, there is a critical need to validate internal control genes. When studying gene expression in bovine polymorphonuclear leukocytes, special attention should be paid to this validation, because polymorphonuclear leukocytes are subjected to numerous physiological influences, depending on the stage of lactation. In this study, 8 commonly used reference genes (ACT, GAPD, H2A, TBP, HPRT1, SDHA, YWHAZ, and 18S rRNA) were evaluated in bovine polymorphonuclear leukocytes. The transcription levels of 6 reference genes were determined using real-time PCR. By geometrically averaging the expression levels of these genes, SDHA, YWHAZ, and 18S rRNA were selected as being the most stable genes for accurate normalization of real-time results of bovine polymorphonuclear leukocytes.
De Ketelaere
A
A
Department of Physiology and Biometrics, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium.
Goossens
K
K
Peelman
L
L
Burvenich
C
C
D000818:Animals; D002417:Cattle / Q000235:genetics*; D005260:Female; D015870:Gene Expression; D020869:Gene Expression Profiling / Q000592:standards / Q000662:veterinary*; D005796:Genes / Q000502:physiology*; D005819:Genetic Markers; D009504:Neutrophils / Q000502:physiology*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary
D005819:Genetic Markers