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Clinical and Vaccine Immunology, September 2008, p. 1369-1373, Vol. 15, No. 9
1071-412X/08/$08.00+0     doi:10.1128/CVI.00074-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Validation of Reference Genes in Cervical Cell Samples from Human Papillomavirus-Infected and -Uninfected Women for Quantitative Reverse Transcription-PCR Assays{triangledown}

Ibrahim I. Daud1,2 and Mark E. Scott3*

Jomo Kenyatta University of Agriculture and Technology, Nairobi, Kenya,1 Infectious Disease Research Training Program, University of California, San Francisco, California 94143,2 Department of Pediatrics, University of California, San Francisco, California, 941433

Received 27 February 2008/ Returned for modification 21 April 2008/ Accepted 2 July 2008

Reference genes for quantitative reverse transcription-PCR (qRT-PCR) studies must be validated for the cell type studied 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, eight candidate genes were screened for amplification efficiency, specificity, and overall stability (by use of geNorm software). The five most stable genes were then further evaluated both for overall stability (geNorm) and intergroup stability (by use of NormFinder software) in specimens from HPV-negative and HPV-positive women. The combination of the glyceraldehyde-3-phosphate dehydrogenase gene (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 showed that these genes represent the most stable two-gene combination, with a NormFinder stability value of 0.130. The fact 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 two genes is likely to be appropriate.

* Corresponding author. Mailing address: University of California, San Francisco, Box 1374, 513 Parnassus Ave., San Francisco, CA 94143-1374. Phone: (415) 476-3260. Fax: (415) 502-1222. E-mail: scottm{at}peds.ucsf.edu

{triangledown} Published ahead of print on 16 July 2008.

Clinical and Vaccine Immunology, September 2008, p. 1369-1373, Vol. 15, No. 9
1071-412X/08/$08.00+0     doi:10.1128/CVI.00074-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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