Data supporting the design and evaluation of a universal primer pair for pseudogene-free amplification of HPRT1 in real-time PCR

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.

Bioinformatics analysis of human and mouse genome were performed to find HPRT1 pseudogenes. HPRT1-specific primers were designed and tested for pseudogene-free amplification of HPRT1 in different cell lines. The primers specificity and efficiency were also determined in qRT-PCR. Data source location Sari, Iran Data accessibility Data is provided with this and the main article [1].

Value of the data
HPRT1 is a sensitive internal control for normalization of gene expression in qRT-PCR. HPSF primer pair allows pseudogene-free amplification of HPRT1 mRNA in qRT-PCR. HPSF primer pair provides specific and high efficient amplification of HPRT1 in qRT-PCR. HPSF primer pair amplifies HPRT1 mRNA across a wide range of mammalian species.
1. Data, experimental design, materials and methods

Primer design
Prior to the identification of human genome sequences, it was shown that HPRT1 contained four pseudogenes on chromosome 3, 5, and 11 [2,3]. Here to identify putative pseudogenes of HPRT1, human and mouse HPRT1 mRNA sequences were used as baits in the Blat and Blast online tools at UCSC Genome Bioinformatics (https://genome.ucsc.edu/) and National Centre for Biotechnology Information (http://www. ncbi.nlm.nih.gov/) respectively. Sequences scored more than 400 and 283 for human and mouse HPRT1 mRNA were selected for further analysis. In consistent with a previous study [4], we also identified three pseudogenes for human HPRT1 on chromosomes 4, 5, and 11 and one for mouse HPRT1 on chromosome 17 (Table 1). To design pseudogene-free primers, HPRT1 mRNA and pseudogene sequences were aligned using CLC Main Workbench software version 5.5 (Qiagen, USA) (see Fig. 1 in Ref. [1]). In order to avoid coamplification of genomic DAN (gDNA) contamination, unique regions of HPRT1 mRNA that corresponded to exonÀ exon junctions were selected. In addition, HPRT1 mRNA sequences of human, mouse, rat, Chinese hamster, golden hamster, and rhesus monkey were aligned to design a universal primer pair capable of amplifying HPRT1 mRNA in these species (Fig. 1). Primers were designed using Allele ID primer design software version 7.5 (Premier Biosoft, USA) ( Table 2). In silico analysis also demonstrated that HPSF primer pair potentially covers a wide taxonomic range in mammals spanning from whale to human (Table 3).

Nucleic acid extraction
The cell lines were cultured to Subconfluent stage in 25 cm 2 culture flasks (SPL, South Korea) and then genomic DNA and total RNA was extracted using AccuPrep s Genomic DNA Extraction Kit (Bioneer, Korea) and Qiagen RNeasy Mini Kit (Qiagen, Germany), respectively. The quantity and integrity of the isolated nucleic acids were verified by Nano-spectrophotometer (WPA, UK) and electrophoresis in a 2% agarose gel, respectively. -a The HPSF primer set is fully matched with these species. b HPSF forward primer contains a mismatch at position of 21 for these species. c HPSF forward primer contains a mismatch at position of 6 for these species.

First strand cDNA synthesis and polymerase chain reaction
Complementary DNA (cDNA) was synthesized from 1 microgram of the total RNA in a 20 ml reaction using the Omniscript-RT-Kit (Qiagen, Germany) according to manufacturer's instruction. PCR reaction was carried out in a total volume of 20 ml containing of 10 mM Tris HCl pH 8.4, 50 mM KCl, 200 nmol each forward and reverse primers, 1.5 mM MgCl 2 , 250 mM dNTP, 1 U of Taq DNA polymerase (Thermo Scientific, Germeny), 2 ml cDNA and 100 ng DNA as templates. The thermal profile included an initial denaturation at 94 1C for 2 min, followed by 40 cycles of amplification at 94 1C for 30 s, 60 1C for 30 s and 72 1C for 30 s and a final extension step at 72 1C for 5 min. The reactions were cycled in an Eppendorf MasterCycler gradient thermal cycler (Germany). The PCR products were visualized after separation on a 2% agarose gel and staining with ethidium bromide. The results of PCR in the all tested cell lines showed that HPSF primers amplified the expected 195 bp products only when cDNA used as templates (see Fig. 1 in Ref. [1]).

DNA sequencing
The accuracy of the amplification reaction was validated by DNA sequencing of the PCR products. The human and mouse amplicons were excised from a 2% agarose gel, and purified using the QIAquick Gel Extraction Kit (Qiagen, Germany) according to the manufacturer's instructions. The quantity of the purified plasmid was then measured by spectrophotometer (Biochrom WPA, UK). About 10 ng of each amplicon was ligated into pTG19-T cloning vector (Vivantis, Malaysia) and transformed into chemically competent bacterial cells. Sequencing of the inserts was performed using capillary DNA analyzer (ABI 3730, Applied Biosystems, USA) after sequencing reactions with a Big Dye Terminator V3.1 Cycle Sequencing Kit (Applied Biosystems). The nucleotide sequences of human and mouse HPRT1 mRNA were submitted to the GenBank database under the accession numbers KR817914 and KR817915, respectively.

Real-time PCR
Real-time PCR reactions were performed using 2X Thermo Scientific Maxima SYBR Green/ROX qPCR Master Mix (Fisher Scientific, Germany) and run in an iCycler iQ5 (Bio RAD, USA) instrument. The reaction was carried out in a total volume of 20 ml containing 10 ml of 2X master mix, 200 nmol of each forward and reverse primer, and 2 ml of 10-fold serially diluted of HeLa cDNA or pTG19/HPRT1 plasmid as templates.
Cycling condition involved an enzyme activation step at 95 1C for 10 min followed by 40 cycles of 95 1C for 10 s, 60 1C for 15 s, and 72 1C for 15 s. In each cycle, an extra step of 72 1C for 10 s was included to collect fluorescence. At the end of PCR, to evaluate specific amplification of the target genes, melting curves ranging from 60 to 95 1C were also included in each run. The slope, intercept, amplification efficiency, and correlation coefficient (R 2 ) of the primer pair were calculated as described previously [5]. The regression line had the correlation coefficient (R 2 value) of 0.999 and the reaction efficiency was calculated 99.5% that confirmed accurate real-time results (Fig. 2A). The melt curve analysis also confirmed the specific amplification of HPRT1 mRNA with no evidence for primer dimer or nonspecific products (Fig. 2B). The importance of pseudogene-free amplification of the housekeeping gene in qRT-PCR has been emphasized for glyceraldehyde-3-phosphate dehydrogenase [6], and the data provided here also indicate that primers should be carefully selected for HPRT1 to ensure accurate transcripts quantification in qRT-PCR.