Identification of Valid Housekeeping Genes for Real-Time Quantitative PCR Analysis of Collapsed Lung Tissues of Neonatal Somatic Cell Nuclear Transfer-Derived Cattle.

Cloned calves produced by somatic cell nuclear transfer frequently suffer alveolar collapse as newborns. To study the underlying pathophysiological mechanisms responsible for this phenomenon, the expression profiles of numerous genes involved in lung development need to be investigated. Quantitative real-time PCR is commonly adopted in gene expression analysis. However, selection of an appropriate reference gene for normalization is critical for obtaining reliable and accurate results. Seven housekeeping genes-β-glucuronidase (GUSB), phosphoglycerate kinase 1 (PGK1), β-2-microglobolin (B2M), peptidylprolyl isomerase A (PPIA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), TATA-box binding protein (TBP), and 5.8S ribosomal RNA (5.8S rRNA)-were selected and evaluated as candidates. Their gene expression levels in the collapsed lungs of deceased neonate cloned calves and normal lung derived from normal calves were assessed. The ranking of gene expression stability was estimated by the geNorm, NormFinder, and BestKeeper programs. 5.8S rRNA and PPIA were determined to be the most stable reference genes by geNorm and BestKeeper, whereas the combination of GAPDH and TBP was suggested as reference genes by NormFinder. Taking these results into account, we conclude that 5.8S rRNA and PPIA could be the most reliable reference genes for studying the genes involved in alveolar collapse. Moreover, 5.8S rRNA could be represented as a uniform reference gene in similar cases.