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Clin. Vaccine Immunol. doi:10.1128/CVI.00074-08
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Validation of Reference Genes for qRT-PCR Assays in Cervical Cell Samples from HPV Infected and Uninfected Women

Jomo Kenyatta University of Agriculture and Technology, Nairobi, Kenya and Infectious Disease Research Training Program, University of California, San Francisco, California, 94143; and Department of Pediatrics, University of California, San Francisco, California, 94143

^* To whom correspondence should be addressed. Email: scottm{at}peds.ucsf.edu.

	Abstract

Reference genes for quantitative (q)RT-PCR studies must be validated for cell type and should be stable between the groups that represent the independent variable in an experimental design. We sought to identify the reference genes in cervical cell specimens showing the most stable expression between human papillomavirus (HPV)-infected and -uninfected women without high-grade cervical intraepithelial neoplasia. Using endocervical cells collected by cytology brush and SYBR Green-based qRT-PCR, 8 candidate genes were screened for amplification efficiency, specificity, and overall stability (by geNorm software). The 5 most stable genes were then further evaluated both for overall stability (geNorm) and intergroup stability (by NormFinder software) in specimens from HPV-negative and HPV-positive women. The combination of GAPDH and RPLP0 was the most stable overall, with a geNorm stability measure of 0.603. The intergroup analysis showed GAPDH to be the most stable single gene and RPLP0 to be second most stable, and also that these genes represent the most stable 2-gene combination, with a NormFinder stability value of 0.130. That these two distinct approaches identified the same pair of genes provides added confidence that—when the focus is on HPV infection—a normalization factor derived from these 2 genes is likely to be appropriate.