Data supporting the design and evaluation of a universal primer pair for pseudogene-free amplification of HPRT1 in real-time PCR
Reza Valadan,
Akbar Hedayatizadeh-Omran,
Mahdyieh Naghavi Alhosseini-Abyazani,
Omolbanin Amjadi,
Alireza Rafiei,
Mohsen Tehrani,
Reza Alizadeh-Navaei
Affiliations
Reza Valadan
Molecular and Cell Biology Research Center (MCBRC), Department of Immunology, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Mazandaran, Iran
Akbar Hedayatizadeh-Omran
Molecular and Cell Biology Research Center (MCBRC), Department of Immunology, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Mazandaran, Iran
Mahdyieh Naghavi Alhosseini-Abyazani
Molecular and Cell Biology Research Center (MCBRC), Department of Immunology, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Mazandaran, Iran
Omolbanin Amjadi
Molecular and Cell Biology Research Center (MCBRC), Department of Immunology, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Mazandaran, Iran
Alireza Rafiei
Molecular and Cell Biology Research Center (MCBRC), Department of Immunology, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Mazandaran, Iran
Mohsen Tehrani
Molecular and Cell Biology Research Center (MCBRC), Department of Immunology, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Mazandaran, Iran
Reza Alizadeh-Navaei
Molecular and Cell Biology Research Center (MCBRC), Department of Immunology, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Mazandaran, Iran
Hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1) is a common housekeeping gene for sample normalization in the quantitative reverse transcriptase polymerase chain (qRT-PCR). However, co-amplification of HPRT1 pseudogenes may affect accurate results obtained in qRT-PCR. We designed a primer pair (HPSF) for pseudogene-free amplification of HPRT1 in qRT-PCR [1]. We showed specific amplification of HPRT1 mRNA in some common laboratory cell lines, including HeLa, NIH/3T3, CHO, BHK, COS-7 and VERO. This article provides data supporting the presence and location of HPRT1 pseudogenes within human and mouse genome, and the strategies used for designing primers that avoid the co-amplification of contaminating pseudogenes in qRT-PCR. In silico analysis of human genome showed three homologous sequences for HPRT1 on chromosomes 4, 5 and 11. The mRNA sequence of HPRT1 was aligned with the pseudogenes, and the primers were designed toward 5′ end of HPRT1 mRNA that was only specific to HPRT1 mRNA not to the pseudogenes. The standard curve plot generated by HPSF primers showed the correlation coefficient of 0.999 and the reaction efficiency of 99.5%. Our findings suggest that HPSF primers can be recommended as a candidate primer pair for accurate and reproducible qRT-PCR assays.