Pseudogene-free amplification of HPRT1 in quantitative reverse transcriptase polymerase chain reaction

Valadan, Reza; Amjadi, Omolbanin; Tehrani, Mohsen; Rafiei, Alireza; Alizadeh‐Navaei, Reza

doi:10.1016/j.ab.2015.05.021

Cited by 11 publications

(12 citation statements)

References 13 publications

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“…1 in Ref. [1] ). In order to avoid co-amplification of genomic DAN (gDNA) contamination, unique regions of HPRT1 mRNA that corresponded to exon−exon junctions were selected.…”

Section: Data Experimental Design Materials and Methodsmentioning

confidence: 91%

“…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.…”

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confidence: 99%

“…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] . …”

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confidence: 99%

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

et al. 2015

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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.

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“…1 in Ref. [1] ). In order to avoid co-amplification of genomic DAN (gDNA) contamination, unique regions of HPRT1 mRNA that corresponded to exon−exon junctions were selected.…”

Section: Data Experimental Design Materials and Methodsmentioning

confidence: 91%

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confidence: 99%

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

et al. 2015

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“…Hprt1 is a common reference gene for normalizing relative expression values in qPcR analysis (40,41). Gapdh is also reliable as a reference gene for quantitative gene expression analysis under experimental conditions (42).…”

Section: Discussionmentioning

confidence: 99%

Selection of reference genes for expression analysis in mouse models of acute alcoholic liver injury

Wang

2018

Int J Mol Med

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Investigations of hepatic gene expression are crucial for determining the molecular factors involved in acute alcoholic liver injury. The results of liver molecular investigations may reveal etiologically important genomic alterations. Therefore, it is necessary to normalize gene expression data to identify stable genes, which may be used as a reference under different experimental conditions. The aim of the present study was to apply reverse transcription‑quantitative polymerase chain reaction analysis and use analysis software to investigate the expression stability of candidate reference genes in hepatic tissues from mice with acute alcoholic liver injury. The acute alcoholic liver injury models were established by the intragastric administration of alcohol (5 mg/kg) in Imprinting Control Region mice. Total RNA was isolated from the mouse livers, following which the expression levels of seven reference genes, β-actin, glyceraldehyde 3-phosphate dehydrogenase (Gadph), glucuronidase β, hypoxanthine phosphoribosyltransferase 1 (Hprt1), 18S ribosomal RNA, TATA binding protein and β‑2 microglobulin, were examined, and gene expression stability was assessed using the geNorm, NormFinder and BestKeeper tools. The geNorm analysis revealed that the gene with the lowest variability was Hprt1. Hprt1 and Gapdh were validated as the optimal reference gene pair in all samples from all groups. The NormFinder and BestKeeper results showed that Hprt1 was the most stable gene in all samples. Alcohol induces endoplasmic reticulum (ER) stress, causing changes in the expression levels of ER stress‑associated genes. The stability of Hprt1 was verified by the expression analysis of ER stress‑associated genes, and gene expression levels in the ethanol groups were upregulated, with a significant difference in expression, compared with those in the control group. Therefore, Hprt1 was selected as the most stable gene, and Hprt1 and Gapdh were determined to be the optimum gene pair in mouse models of acute alcoholic liver injury. The reliability of the Hprt1 gene was confirmed by expression analysis of ER stress‑associated genes.

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“…PCR array results were analysed with Rotor‐Gene 6000 series software (Corbett Research, Mortlake, NSW, Australia). Samples were normalised to the HPRT1 housekeeper gene with an assay batch known not to react to HPRT pseudogenes . Data analysis utilised GraphPad Prism version 5.0b (GraphPad Software Inc., La Jolla, CA, USA) with the nonparametric Mann‐Whitney U test for statistical analysis.…”

Section: Methodsmentioning

confidence: 99%

Does gene expression in laryngeal subsites differ between patients with laryngopharyngeal reflux and controls?

Wood

Hussey

Woods

et al. 2017

Clinical Otolaryngology

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Objective: To identify laryngeal mRNA gene changes in patients with laryngopharyngeal reflux (LPR).Method: Laryngeal biopsies from non-smoking LPR patients (n=10; Reflux Symptom Index (RSI) >12 and a Reflux Finding Score (RFS) >6) and controls (n=9; RSI <12 and RFS <6) were collected from four subsites (true vocal cord, false vocal cord, medial arytenoid and posterior commissure) of the larynx. qRT-PCR analyses were conducted on 20 reflux-and inflammation-related genes, including interleukins 6 and 8, cytokeratins 8 and 14, mucin genes MUC1, MUC2, MUC3B, MUC4, MUC5B, MUC6 and MUC7 and carbonic anhydrase III. Statistical analysis (Mann-Whitney U test) compared gene expression levels between LPR and control groups at each subsite.Results: Site-specific differences in squamous metaplasia and gene expression were noted in LPR patients, with the majority present in the medial arytenoid region. Significant.differences were noted in genes related to mucosal defence and inflammation, including CRNN, CD1d, TGFb-1, MUC2, MUC5B and CDH1.Conclusion: Whilst the posterior commissure is commonly identified as the area demonstrating the most significant macroscopic change in LPR, the histological changes and genes assessed here showed more pronounced LPR associated differences in the medial arytenoid. We identified differences in expression of mucin genes, cytokeratin-14 and molecular markers of inflammation. Whilst some of these changes may be metaplasia-related, further evaluation of the mRNA expression of these genes may provide a useful biomarker panel for diagnosis and therapeutic monitoring of LPR.

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Pseudogene-free amplification of HPRT1 in quantitative reverse transcriptase polymerase chain reaction

Cited by 11 publications

References 13 publications

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

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

Selection of reference genes for expression analysis in mouse models of acute alcoholic liver injury

Does gene expression in laryngeal subsites differ between patients with laryngopharyngeal reflux and controls?

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