Accepted Manuscript Notes & Tips Pseudogene-free amplification of HPRT1 in qRT-PCR Reza Valadan, Omolbanin Amjadi, Mohsen Tehrani, Alireza Rafiei, Akbar Hedayatizadeh-Omran, Reza Alizadeh-Navaei PII: DOI: Reference:
S0003-2697(15)00289-4 http://dx.doi.org/10.1016/j.ab.2015.05.021 YABIO 12095
To appear in:
Analytical Biochemistry
Received Date: Revised Date: Accepted Date:
1 September 2014 28 May 2015 31 May 2015
Please cite this article as: R. Valadan, O. Amjadi, M. Tehrani, A. Rafiei, A. Hedayatizadeh-Omran, R. AlizadehNavaei, Pseudogene-free amplification of HPRT1 in qRT-PCR, Analytical Biochemistry (2015), doi: http:// dx.doi.org/10.1016/j.ab.2015.05.021
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Pseudogene-free amplification of HPRT1 in qRT-PCR Reza Valadan1¶, Omolbanin Amjadi1¶, Mohsen Tehrani1,2, Alireza Rafiei1*, Akbar Hedayatizadeh-Omran1, Reza Alizadeh-Navaei1 1-Molecular and Cell Biology Research Center (MCBRC), Department of Immunology, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Mazandaran, Iran 2- Department of Immunology, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Mazandaran, Iran *Corresponding author: Alireza Rafiei PhD in Immunology, Professor. Email: ([email protected]) ¶ These authors contributed equally to this work.
Abstract: Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) provides a powerful tool for precise gene expression analysis. The accuracy of the results highly depends upon careful selection of a reference gene for data normalization. Hypoxanthine-guanine phosphoribosyl transferase 1 (HPRT1) is a frequently used housekeeping gene for normalizing relative expression values. However, the existence of processed pseudogenes for HPRT1 might interfere with reliable results obtained in qRT-PCR due to amplification of unintended products. Herein, we designed a primer pair for pseudogene-free amplification of HPRT1 in qRT-PCR. We demonstrated that this primer pair specifically amplified HPRT1 mRNA sequence while avoiding co-amplification of the pseudogenes. Key words: HPRT1, Pseudogenes, qRT-PCR, Reference gene
1
Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) provides a sensitive method to detect gene expression levels even in small amounts of samples [1]. A constitutively expressed housekeeping gene is usually used as a reference gene to normalize the effect of variability in cell number, amount of extracted RNA, specificity of primers, and the presence of PCR inhibitors in samples [2, 3]. However, most of currently used housekeeping genes have variable expression levels in different tissues. Therefore, an ideal housekeeping gene should be carefully selected according to the type of tissues and the treatments [4]. Hypoxanthine phosphoribosyl transferase-1 (HPRT1) is a commonly used internal control in qRT-PCR and has been recommended as a sensitive housekeeping gene, especially when low-abundance transcripts are to be investigated [5]. However, the presence of processed pseudogenes for HPRT1 in the genome [6] may affect the use of HPRT1 as a valid internal control in qRT-PCR [7]. Pseudogenes are similar to functional genes in sequence while they are non-translatable. A high nucleotide sequence similarity between the pseudogenes and HPRT1 mRNA leads to coamplification of pseudogenes in qRT-PCR especially when genomic DNA (gDNA) contamination is probable [8]. The amplified PCR products have the same size thus are not distinguishable [9]. DNaseI treatment is a proposed method used to remove residual DNA yet; the procedure cannot guarantee complete removal contaminating DNA, as well as the original quality of extracted RNA [10]. Moreover, transcriptionally active pseudogenes might be transcripted into mRNA and subsequently converted into cDNA during reverse transcription. This, in turn, leads to co-amplification of pseudogenes in the real-time PCR. Here, to amplify pseudogene-free sequences of HPRT1 in real-time PCR, we designed a primer pair in a way that specifically amplifies HPRT1 mRNA sequence while avoiding co-amplification of the pseudogenes or gDNA. To do this, human and mouse HPRT1 mRNA homologous sequences 2
were identified using discontiguous megablast and blastn at National Centre for Biotechnology Information (NCBI) (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Sequences scored more than 400 and had more than 75% identity with the human HPRT1 mRNA were selected for further analysis. The same criteria were considered for mouse sequences. Three goals were considered at the same time for the primer design; 1- avoiding mis-priming to gDNA, 2- avoiding mis-priming to the pseudogenes, and 3- amplification of HPRT1 mRNA sequences in commonly used laboratory animals. Therefore, HPRT1 mRNA and pseudogene sequences were aligned using CLC Main Workbench software (Qiagen, USA) (Figure-1). Unique regions of HPRT1 mRNA corresponding to exon-exon junctions were selected for primer design. Additionally, HPRT1 mRNA sequences of human, mouse, rat, Chinese and golden hamster, and rhesus monkey were aligned to design a universal primer pair capable of amplifying HPRT1 mRNA across these species. Primers were designed using Allele ID primer design software version 7.5 (Premier Biosoft, USA). The primer pair designated HPSF in this paper (HPSF-F: 5´GGACTAATTATGGACAGGACTG-`3 and HPSF-R: 5´GCTCTTCAGTCTGATAAAATCTAC-`3) The results of BLAST showed four homologous sequences for human HPRT1 mRNA on chromosomes 4, 5, and 11. HPRTP1 on chromosome 4 consists of two exon-like sequences separated by a short 61 bp intron. HPRTP2 is located on chromosome 5 spanning 1440 bp with the highest coverage and identity to HPRT1 mRNA. HPRTP3 and HPRTP4 are located on chromosome 11 separated by an 8544 bp sequence. Similarly, results of BLAST for mouse HPRT1 mRNA showed a 765 homologous sequence located on mouse chromosome 17. Alignment of mouse HPRT1 mRNA sequence with the putative pseudogene on chromosome 17 showed no overlapping region between the first 525 bp of mouse HPRT1 mRNA and HPRT1 3
pseudogene, therefore, this region was unique to mouse HPRT1 mRNA (alignment not shown). Prior to the identification of human genome sequences, it was shown that HPRT1 contained 4 pseudogenes on chromosome 3, 5, and 11. Southern blot analysis of human cells indicated that two of these sequences were localized on chromosome11, one on chromosome 3, and another on chromosome 5 [11]. This is consistent with the results reported by Nicklas in 2006, suggesting a unique 7.2 kb insert on chromosome 11, which could be attributed to two separated pseudogenes (HPRTP3 and HPRTP4) [6]. However, more recently, in silico analysis of human genome showed that HPRT1 included 3 pseudogenes on chromosome 4, 5, and 11 [12]. Although HPRTP3 and HPRTP4 are listed as two separated pseudogenes at NCBI, it is not known whether they are originally a unique unprocessed pseudogene with an intervening 8544bp intron or they are two separated pseudogenes on chromosome 11. Next, to show that HPSF primers specifically amplify pseudogene-free sequences of human HPRT1 mRNA as well as some other widely used laboratory species, we performed separate PCR using cDNA and gDNA as templates. Genomic DNA and total RNA were extracted from exponentially growing HeLa, NIH/3T3, CHO, BHK, VERO, and COS-7 cell lines using AccuPrep®Genomic DNA Extraction Kit (Bioneeer, Korea) and Qiagen RNeasy Mini Kit (Qiagen, Germany), respectively. These cell lines are originated from human, mouse, Chinese and golden hamster and green monkey. One microgram of total RNA was then reverse transcribed into cDNA using the Omniscript-RT-Kit (Qiagen, Germany) in accordance with manufacture’s instruction. PCR was carried out in a total volume of 20 µL containing of 10 mM Tris HCl pH 8.4, 50 mM KCl, 200 nmol each forward and reverse primers, 1.5 mM MgCl2, 250 µM dNTP, 1 U of Taq DNA polymerase (Thermo Scientific, Germeny), 2 µL cDNA and 100 ng DNA as template. Amplifications were carried out in an Eppendorf Master Cycler gradient 4
thermal cycler (Germany). The results of PCR showed that HPSF primers amplified a 195 bp product only when cDNA used as template (Figure-2). In addition, we showed that HPSF primers specifically amplified HPRT1 mRNA sequences in all selected species (Figure-2). Furthermore, in silico analysis of HPSF primers also demonstrated that this primer set is not only target-specific to human and mouse HPRT1 sequences, but also may potentially bind to HPRT1 mRNA sequences from several other species. Next, we aimed to determine the efficiency of the HPSF primers in qRT-PCR using the standards of 10-fold serial dilutions of HeLa cDNA. qRT-PCR was carried out using Thermo Scientific Maxima SYBR Green qPCR Master Mix (Fisher Scientific, Germany), 200 nmol of each forward and reverse primer, and 2 µL of 10-fold serially diluted of HeLa cDNA as templates. Amplifications were performed using iCycler iQ5 (Bio RAD, USA) real-time PCR machine. Based on the logarithmic serial dilutions of HeLa cDNA, a standard curve was generated. The slope, intercept, amplification efficiency, and correlation coefficient (R2) of the primer pair were calculated. The results showed that the regression line had a correlation coefficient (R2 value) of 0.99. The reaction efficiency was then calculated 99.5% that confirmed accurate real-time results. Two sets of primers have been previously designed for human (HPRT1) and mouse (Hprt). It was shown that both primer pairs were specific for human and mouse HPRT1 mRNA sequences, but not for pseudogenes. However, the derived amplicons were rather long, 561 bp and 448 bp for HPRT1 and Hprt, respectively, which might significantly reduce PCR efficiency in the real time PCR, therefore, inappropriate for qRT-PCR studies [13]. In summary, we designed and evaluate a universal primer set for selective amplification of HPRT1 mRNA sequence in qRT-PCR. Moreover, we also showed that this primer pair amplified HPRT1 mRNA sequences in several laboratory species experimentally, and potentially covers a 5
wide taxonomic range in mammals spanning from whale to human. Therefore, our findings suggest that HPSF primers can be recommended as a candidate universal primer set for accurate and reproducible qRT-PCR assays. Acknowledgments This study was supported by the Molecular and Cell Biology Research Center (MCBRC), Mazandaran University of Medical Sciences, Sari, Mazandaran, Iran and a grant from Iranian Nation Science Foundation (INSF) (91002930). References: [1] L. Fink, W. Seeger, L. Ermert, J. Hanze, U. Stahl, F. Grimminger, W. Kummer, R.M. Bohle, Real-time quantitative RT-PCR after laser-assisted cell picking, Nat Med 4 (1998) 1329-1333. [2] A. Turabelidze, S. Guo, L.A. DiPietro, Importance of housekeeping gene selection for accurate reverse transcription-quantitative polymerase chain reaction in a wound healing model, Wound Repair Regen. 18 (2010) 460-466. [3] Z. Zhai, Y. Yao, Y. Wang, Importance of suitable reference gene selection for quantitative RT-PCR during ATDC5 cells chondrocyte differentiation, PLoS One. 8 (2013) e64786. [4] I. Chervoneva, Y. Li, S. Schulz, S. Croker, C. Wilson, S.A. Waldman, T. Hyslop, Selection of optimal reference genes for normalization in quantitative RT-PCR, BMC Bioinformatics. 11 (2010) 253. [5] X. Zhang, L. Ding, A.J. Sandford, Selection of reference genes for gene expression studies in human neutrophils by real-time PCR, BMC Mol Biol. 6 (2005) 4. [6] J.A. Nicklas, Pseudogenes of the human HPRT1 gene, Environ Mol Mutagen. 47 (2006) 212218.
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[7] L.N. Sellner, G.R. Turbett, The presence of a pseudogene may affect the use of HPRT as an endogenous mRNA control in RT-PCR, Mol Cell Probes. 10 (1996) 481-483. [8] P. Tschentscher, C. Wagener, M. Neumaier, Sensitive and specific cytokeratin 18 reverse transcription-polymerase chain reaction that excludes amplification of processed pseudogenes from contaminating genomic DNA, Clin Chem 43 (1997) 2244-2250. [9] K.A. Kreuzer, U. Lass, O. Landt, A. Nitsche, J. Laser, H. Ellerbrok, G. Pauli, D. Huhn, C.A. Schmidt, Highly sensitive and specific fluorescence reverse transcription-PCR assay for the pseudogene-free detection of beta-actin transcripts as quantitative reference, Clin Chem 45 (1999) 297-300. [10] P.A. Rochelle, A.J. Weightman, J.C. Fry, DNase I treatment of Taq DNA polymerase for complete PCR decontamination, Biotechniques 13 (1992) 520. [11] P.I. Patel, R.L. Nussbaum, P.E. gramson, D.H. Ledbetter, C.T. Caskey, A.C. Chinault, Organization of the HPRT gene and related sequences in the human genome, Somat Cell Mol Genet 10 (1984) 483-493. [12] Y. Sun, Y. Li, D. Luo, D.J. Liao, Pseudogenes as weaknesses of ACTB (Actb) and GAPDH (Gapdh) used as reference genes in reverse transcription and polymerase chain reactions, PLoS One 7 (2012) e41659. Figure legends: Figure-1 Multiple alignment of HPRT1 mRNA with HPRTP1-3 sequences to identify appropriate regions for primer design. Low level of sequence similarity between HPRT1 mRNA and HPRTPs at exon2 and exon3 junction provided a unique site for primer design. Black boxes show forward and reverse primer binding sites. Red box shows 3´ mismatch at the forward primer binding site. TG bases are only 7
present at 3′ end of the forward primer binding site, enabling pseudogene-free amplification of HPRT1 mRNA sequence in qRT-PCR. Figure-2 Pseudogene-free amplification of HPRT1 mRNA sequences by HPSF primer set in different species. HPSF primers amplified a 195 bp product only when cDNA used as template. The selected cell lines represent some commonly used laboratory species. M: DNA ladder.
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S0003-2697(15)00289-4 http://dx.doi.org/10.1016/j.ab.2015.05.021 YABIO 12095
To appear in:
Analytical Biochemistry
Received Date: Revised Date: Accepted Date:
1 September 2014 28 May 2015 31 May 2015
Please cite this article as: R. Valadan, O. Amjadi, M. Tehrani, A. Rafiei, A. Hedayatizadeh-Omran, R. AlizadehNavaei, Pseudogene-free amplification of HPRT1 in qRT-PCR, Analytical Biochemistry (2015), doi: http:// dx.doi.org/10.1016/j.ab.2015.05.021
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Pseudogene-free amplification of HPRT1 in qRT-PCR Reza Valadan1¶, Omolbanin Amjadi1¶, Mohsen Tehrani1,2, Alireza Rafiei1*, Akbar Hedayatizadeh-Omran1, Reza Alizadeh-Navaei1 1-Molecular and Cell Biology Research Center (MCBRC), Department of Immunology, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Mazandaran, Iran 2- Department of Immunology, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Mazandaran, Iran *Corresponding author: Alireza Rafiei PhD in Immunology, Professor. Email: ([email protected]) ¶ These authors contributed equally to this work.
Abstract: Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) provides a powerful tool for precise gene expression analysis. The accuracy of the results highly depends upon careful selection of a reference gene for data normalization. Hypoxanthine-guanine phosphoribosyl transferase 1 (HPRT1) is a frequently used housekeeping gene for normalizing relative expression values. However, the existence of processed pseudogenes for HPRT1 might interfere with reliable results obtained in qRT-PCR due to amplification of unintended products. Herein, we designed a primer pair for pseudogene-free amplification of HPRT1 in qRT-PCR. We demonstrated that this primer pair specifically amplified HPRT1 mRNA sequence while avoiding co-amplification of the pseudogenes. Key words: HPRT1, Pseudogenes, qRT-PCR, Reference gene
1
Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) provides a sensitive method to detect gene expression levels even in small amounts of samples [1]. A constitutively expressed housekeeping gene is usually used as a reference gene to normalize the effect of variability in cell number, amount of extracted RNA, specificity of primers, and the presence of PCR inhibitors in samples [2, 3]. However, most of currently used housekeeping genes have variable expression levels in different tissues. Therefore, an ideal housekeeping gene should be carefully selected according to the type of tissues and the treatments [4]. Hypoxanthine phosphoribosyl transferase-1 (HPRT1) is a commonly used internal control in qRT-PCR and has been recommended as a sensitive housekeeping gene, especially when low-abundance transcripts are to be investigated [5]. However, the presence of processed pseudogenes for HPRT1 in the genome [6] may affect the use of HPRT1 as a valid internal control in qRT-PCR [7]. Pseudogenes are similar to functional genes in sequence while they are non-translatable. A high nucleotide sequence similarity between the pseudogenes and HPRT1 mRNA leads to coamplification of pseudogenes in qRT-PCR especially when genomic DNA (gDNA) contamination is probable [8]. The amplified PCR products have the same size thus are not distinguishable [9]. DNaseI treatment is a proposed method used to remove residual DNA yet; the procedure cannot guarantee complete removal contaminating DNA, as well as the original quality of extracted RNA [10]. Moreover, transcriptionally active pseudogenes might be transcripted into mRNA and subsequently converted into cDNA during reverse transcription. This, in turn, leads to co-amplification of pseudogenes in the real-time PCR. Here, to amplify pseudogene-free sequences of HPRT1 in real-time PCR, we designed a primer pair in a way that specifically amplifies HPRT1 mRNA sequence while avoiding co-amplification of the pseudogenes or gDNA. To do this, human and mouse HPRT1 mRNA homologous sequences 2
were identified using discontiguous megablast and blastn at National Centre for Biotechnology Information (NCBI) (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Sequences scored more than 400 and had more than 75% identity with the human HPRT1 mRNA were selected for further analysis. The same criteria were considered for mouse sequences. Three goals were considered at the same time for the primer design; 1- avoiding mis-priming to gDNA, 2- avoiding mis-priming to the pseudogenes, and 3- amplification of HPRT1 mRNA sequences in commonly used laboratory animals. Therefore, HPRT1 mRNA and pseudogene sequences were aligned using CLC Main Workbench software (Qiagen, USA) (Figure-1). Unique regions of HPRT1 mRNA corresponding to exon-exon junctions were selected for primer design. Additionally, HPRT1 mRNA sequences of human, mouse, rat, Chinese and golden hamster, and rhesus monkey were aligned to design a universal primer pair capable of amplifying HPRT1 mRNA across these species. Primers were designed using Allele ID primer design software version 7.5 (Premier Biosoft, USA). The primer pair designated HPSF in this paper (HPSF-F: 5´GGACTAATTATGGACAGGACTG-`3 and HPSF-R: 5´GCTCTTCAGTCTGATAAAATCTAC-`3) The results of BLAST showed four homologous sequences for human HPRT1 mRNA on chromosomes 4, 5, and 11. HPRTP1 on chromosome 4 consists of two exon-like sequences separated by a short 61 bp intron. HPRTP2 is located on chromosome 5 spanning 1440 bp with the highest coverage and identity to HPRT1 mRNA. HPRTP3 and HPRTP4 are located on chromosome 11 separated by an 8544 bp sequence. Similarly, results of BLAST for mouse HPRT1 mRNA showed a 765 homologous sequence located on mouse chromosome 17. Alignment of mouse HPRT1 mRNA sequence with the putative pseudogene on chromosome 17 showed no overlapping region between the first 525 bp of mouse HPRT1 mRNA and HPRT1 3
pseudogene, therefore, this region was unique to mouse HPRT1 mRNA (alignment not shown). Prior to the identification of human genome sequences, it was shown that HPRT1 contained 4 pseudogenes on chromosome 3, 5, and 11. Southern blot analysis of human cells indicated that two of these sequences were localized on chromosome11, one on chromosome 3, and another on chromosome 5 [11]. This is consistent with the results reported by Nicklas in 2006, suggesting a unique 7.2 kb insert on chromosome 11, which could be attributed to two separated pseudogenes (HPRTP3 and HPRTP4) [6]. However, more recently, in silico analysis of human genome showed that HPRT1 included 3 pseudogenes on chromosome 4, 5, and 11 [12]. Although HPRTP3 and HPRTP4 are listed as two separated pseudogenes at NCBI, it is not known whether they are originally a unique unprocessed pseudogene with an intervening 8544bp intron or they are two separated pseudogenes on chromosome 11. Next, to show that HPSF primers specifically amplify pseudogene-free sequences of human HPRT1 mRNA as well as some other widely used laboratory species, we performed separate PCR using cDNA and gDNA as templates. Genomic DNA and total RNA were extracted from exponentially growing HeLa, NIH/3T3, CHO, BHK, VERO, and COS-7 cell lines using AccuPrep®Genomic DNA Extraction Kit (Bioneeer, Korea) and Qiagen RNeasy Mini Kit (Qiagen, Germany), respectively. These cell lines are originated from human, mouse, Chinese and golden hamster and green monkey. One microgram of total RNA was then reverse transcribed into cDNA using the Omniscript-RT-Kit (Qiagen, Germany) in accordance with manufacture’s instruction. PCR was carried out in a total volume of 20 µL containing of 10 mM Tris HCl pH 8.4, 50 mM KCl, 200 nmol each forward and reverse primers, 1.5 mM MgCl2, 250 µM dNTP, 1 U of Taq DNA polymerase (Thermo Scientific, Germeny), 2 µL cDNA and 100 ng DNA as template. Amplifications were carried out in an Eppendorf Master Cycler gradient 4
thermal cycler (Germany). The results of PCR showed that HPSF primers amplified a 195 bp product only when cDNA used as template (Figure-2). In addition, we showed that HPSF primers specifically amplified HPRT1 mRNA sequences in all selected species (Figure-2). Furthermore, in silico analysis of HPSF primers also demonstrated that this primer set is not only target-specific to human and mouse HPRT1 sequences, but also may potentially bind to HPRT1 mRNA sequences from several other species. Next, we aimed to determine the efficiency of the HPSF primers in qRT-PCR using the standards of 10-fold serial dilutions of HeLa cDNA. qRT-PCR was carried out using Thermo Scientific Maxima SYBR Green qPCR Master Mix (Fisher Scientific, Germany), 200 nmol of each forward and reverse primer, and 2 µL of 10-fold serially diluted of HeLa cDNA as templates. Amplifications were performed using iCycler iQ5 (Bio RAD, USA) real-time PCR machine. Based on the logarithmic serial dilutions of HeLa cDNA, a standard curve was generated. The slope, intercept, amplification efficiency, and correlation coefficient (R2) of the primer pair were calculated. The results showed that the regression line had a correlation coefficient (R2 value) of 0.99. The reaction efficiency was then calculated 99.5% that confirmed accurate real-time results. Two sets of primers have been previously designed for human (HPRT1) and mouse (Hprt). It was shown that both primer pairs were specific for human and mouse HPRT1 mRNA sequences, but not for pseudogenes. However, the derived amplicons were rather long, 561 bp and 448 bp for HPRT1 and Hprt, respectively, which might significantly reduce PCR efficiency in the real time PCR, therefore, inappropriate for qRT-PCR studies [13]. In summary, we designed and evaluate a universal primer set for selective amplification of HPRT1 mRNA sequence in qRT-PCR. Moreover, we also showed that this primer pair amplified HPRT1 mRNA sequences in several laboratory species experimentally, and potentially covers a 5
wide taxonomic range in mammals spanning from whale to human. Therefore, our findings suggest that HPSF primers can be recommended as a candidate universal primer set for accurate and reproducible qRT-PCR assays. Acknowledgments This study was supported by the Molecular and Cell Biology Research Center (MCBRC), Mazandaran University of Medical Sciences, Sari, Mazandaran, Iran and a grant from Iranian Nation Science Foundation (INSF) (91002930). References: [1] L. Fink, W. Seeger, L. Ermert, J. Hanze, U. Stahl, F. Grimminger, W. Kummer, R.M. Bohle, Real-time quantitative RT-PCR after laser-assisted cell picking, Nat Med 4 (1998) 1329-1333. [2] A. Turabelidze, S. Guo, L.A. DiPietro, Importance of housekeeping gene selection for accurate reverse transcription-quantitative polymerase chain reaction in a wound healing model, Wound Repair Regen. 18 (2010) 460-466. [3] Z. Zhai, Y. Yao, Y. Wang, Importance of suitable reference gene selection for quantitative RT-PCR during ATDC5 cells chondrocyte differentiation, PLoS One. 8 (2013) e64786. [4] I. Chervoneva, Y. Li, S. Schulz, S. Croker, C. Wilson, S.A. Waldman, T. Hyslop, Selection of optimal reference genes for normalization in quantitative RT-PCR, BMC Bioinformatics. 11 (2010) 253. [5] X. Zhang, L. Ding, A.J. Sandford, Selection of reference genes for gene expression studies in human neutrophils by real-time PCR, BMC Mol Biol. 6 (2005) 4. [6] J.A. Nicklas, Pseudogenes of the human HPRT1 gene, Environ Mol Mutagen. 47 (2006) 212218.
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[7] L.N. Sellner, G.R. Turbett, The presence of a pseudogene may affect the use of HPRT as an endogenous mRNA control in RT-PCR, Mol Cell Probes. 10 (1996) 481-483. [8] P. Tschentscher, C. Wagener, M. Neumaier, Sensitive and specific cytokeratin 18 reverse transcription-polymerase chain reaction that excludes amplification of processed pseudogenes from contaminating genomic DNA, Clin Chem 43 (1997) 2244-2250. [9] K.A. Kreuzer, U. Lass, O. Landt, A. Nitsche, J. Laser, H. Ellerbrok, G. Pauli, D. Huhn, C.A. Schmidt, Highly sensitive and specific fluorescence reverse transcription-PCR assay for the pseudogene-free detection of beta-actin transcripts as quantitative reference, Clin Chem 45 (1999) 297-300. [10] P.A. Rochelle, A.J. Weightman, J.C. Fry, DNase I treatment of Taq DNA polymerase for complete PCR decontamination, Biotechniques 13 (1992) 520. [11] P.I. Patel, R.L. Nussbaum, P.E. gramson, D.H. Ledbetter, C.T. Caskey, A.C. Chinault, Organization of the HPRT gene and related sequences in the human genome, Somat Cell Mol Genet 10 (1984) 483-493. [12] Y. Sun, Y. Li, D. Luo, D.J. Liao, Pseudogenes as weaknesses of ACTB (Actb) and GAPDH (Gapdh) used as reference genes in reverse transcription and polymerase chain reactions, PLoS One 7 (2012) e41659. Figure legends: Figure-1 Multiple alignment of HPRT1 mRNA with HPRTP1-3 sequences to identify appropriate regions for primer design. Low level of sequence similarity between HPRT1 mRNA and HPRTPs at exon2 and exon3 junction provided a unique site for primer design. Black boxes show forward and reverse primer binding sites. Red box shows 3´ mismatch at the forward primer binding site. TG bases are only 7
present at 3′ end of the forward primer binding site, enabling pseudogene-free amplification of HPRT1 mRNA sequence in qRT-PCR. Figure-2 Pseudogene-free amplification of HPRT1 mRNA sequences by HPSF primer set in different species. HPSF primers amplified a 195 bp product only when cDNA used as template. The selected cell lines represent some commonly used laboratory species. M: DNA ladder.
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