Microarray-driven validation of reference genes for quantitative real-time polymerase chain reaction in a rat vocal fold model of mucosal injury.
Relative quantification by normalization against a stably expressed reference gene is a widely used data analysis method in microarray and quantitative real-time polymerase chain reaction (qRT-PCR) platforms; however, recent evidence suggests that many commonly utilized reference genes are unstable in certain experimental systems and situations. The primary aim of this study, therefore, was to screen and identify stably expressed reference genes in a well-established rat model of vocal fold mucosal injury. We selected and evaluated the expression stability of nine candidate reference genes. Ablim1, Sptbn1, and Wrnip1 were identified as stably expressed in a model-specific microarray dataset and were further validated as suitable reference genes in an independent qRT-PCR experiment using 2(-DeltaCT) and pairwise comparison-based (geNorm) analyses. Parallel analysis of six commonly used reference genes identified Sdha as the only stably expressed candidate in this group. Sdha, Sptbn1, and the geometric mean of Sdha and Sptbn1 each provided accurate normalization of target gene Tgfb1; Gapdh, the least stable candidate gene in our dataset, provided inaccurate normalization and an invalid experimental result. The stable reference genes identified here are suitable for accurate normalization of target gene expression in vocal fold mucosal injury experiments.
Chang
Zhen
Z
Division of Otolaryngology, Department of Surgery, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA.
Ling
Changying
C
Yamashita
Masaru
M
Welham
Nathan V
NV
D000818:Animals; D004195:Disease Models, Animal; D005786:Gene Expression Regulation; D007249:Inflammation / Q000235:genetics; D008297:Male; D009092:Mucous Membrane / Q000293:injuries* / Q000378:metabolism* / Q000473:pathology; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D051381:Rats; D011916:Rats, Inbred F344; D012015:Reference Standards; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012984:Software; D053773:Transforming Growth Factor beta1 / Q000235:genetics / Q000378:metabolism; D014827:Vocal Cords / Q000293:injuries* / Q000378:metabolism* / Q000473:pathology; D014945:Wound Healing / Q000235:genetics
C508962:Tgfb1 protein, rat; D053773:Transforming Growth Factor beta1