Microarray-driven validation of reference genes for quantitative real-time polymerase chain reaction in a rat vocal fold model of mucosal injury

Division of Otolaryngology, Department of Surgery, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA.
Analytical Biochemistry (Impact Factor: 2.22). 11/2010; 406(2):214-21. DOI: 10.1016/j.ab.2010.07.021
Source: PubMed


Relative quantification by normalization against a stably expressed reference gene is a widely used data analysis method in microarray and quantitative real-time polymerase chain reaction (qRT-PCR) platforms; however, recent evidence suggests that many commonly utilized reference genes are unstable in certain experimental systems and situations. The primary aim of this study, therefore, was to screen and identify stably expressed reference genes in a well-established rat model of vocal fold mucosal injury. We selected and evaluated the expression stability of nine candidate reference genes. Ablim1, Sptbn1, and Wrnip1 were identified as stably expressed in a model-specific microarray dataset and were further validated as suitable reference genes in an independent qRT-PCR experiment using 2(-DeltaCT) and pairwise comparison-based (geNorm) analyses. Parallel analysis of six commonly used reference genes identified Sdha as the only stably expressed candidate in this group. Sdha, Sptbn1, and the geometric mean of Sdha and Sptbn1 each provided accurate normalization of target gene Tgfb1; Gapdh, the least stable candidate gene in our dataset, provided inaccurate normalization and an invalid experimental result. The stable reference genes identified here are suitable for accurate normalization of target gene expression in vocal fold mucosal injury experiments.

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Available from: Masaru Yamashita, Feb 19, 2015
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    • "The majority of gene expression studies on preimplantation embryos have been performed using only one housekeeping gene [63–65]. Contrary to our results, Chang et al. [66] reported significantly lower stability values for four reference genes (sdha, sptbn, ablim and wrnip); for example, sptbn had a higher stability value in our experiments. The differences in expression stability may be the result of different media or developmental stages analyzed in the compared experiments. "
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    ABSTRACT: Background Real-time quantitative reverse-transcriptase polymerase chain reaction (RT-qPCR) is the most sensitive, and valuable technique for rare mRNA detection. However, the expression profiles of reference genes under different experimental conditions, such as different mouse strains, developmental stage, and culture conditions have been poorly studied. Results mRNA stability of the actb, gapdh, sdha, ablim, ywhaz, sptbn, h2afz, tgfb1, 18 s and wrnip genes was analyzed. Using the NormFinder program, the most stable genes are as follows: h2afz for the B6D2F-1 and C57BL/6 strains; sptbn for ICR; h2afz for KOSOM and CZB cultures of B6D2F-1 and C57BL/6 strain-derived embryos; wrnip for M16 culture of B6D2F-1 and C57BL/6 strain-derived embryos; ywhaz, tgfb1, 18 s, 18 s, ywhaz, and h2afz for zygote, 2-cell, 4-cell, 8-cell, molular, and blastocyst embryonic stages cultured in KSOM medium, respectively; h2afz, wrnip, wrnip, h2afz, ywhaz, and ablim for zygote, 2-cell, 4-cell, 8-cell, molular, and blastocyst stage embryos cultured in CZB medium, respectively; 18 s, h2afz, h2afz, actb, h2afz, and wrnip for zygote, 2-cell, 4-cell, 8-cell, molular, and blastocyst stage embryos cultured in M16 medium, respectively. Conclusions These results demonstrated that candidate reference genes for normalization of target gene expression using RT-qPCR should be selected according to mouse strains, developmental stage, and culture conditions. Electronic supplementary material The online version of this article (doi:10.1186/1756-0500-7-675) contains supplementary material, which is available to authorized users.
    BMC Research Notes 09/2014; 7(1):675. DOI:10.1186/1756-0500-7-675
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    • "These results indicated that the reference genes we chose for gene expression quantification in preimplantation development were appropriate. Moreover, latest studies also revealed that normalization of target gene using unstable reference genes led to significantly different results compared with those using suitable reference genes [24]–[26]. Unfortunately, a number of studies still use traditional reference genes, such asβ-actin and Gapdh, or selected a single randomly gene for the normalization of gene expression, and these choices are likely to impair the accuracy of the result [27]–[31]. Therefore, selection of appropriate reference genes is critical to ensure the accuracy of target gene expression quantification using qPCR experiments. "
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    ABSTRACT: Real-time reverse transcription quantitative polymerase chain reaction (qPCR) has become the most frequently used system for studies of gene expression. Manystudies have provided reliable evidence that the transcription levels of reference genes are not constant at different developmental stages and in different experimental conditions. However, suitable reference genes which are stably expressed in polyploid preimplantation embryos of different developmental stages have not yet been identified. Therefore, it is critical to verify candidate reference genes to analyze gene expression accurately in both diploid and polyploid embryos. We examined the expression levels of 12 candidate reference genes in preimplantation embryos of four different ploidies at six developmental stages. Stability analysis of the reference genes was performed by four independent software programs, and the stability of three genes was evaluated by comparison with the Oct4 expression level during preimplantation development in diploid embryos. The expression levels of most genes in the polyploid embryos were higher than that in the diploid embryos, but the increasing degree were disproportionate with the ploidies. There were no significant difference in reference gene expressions among embryos of different ploidies when they reached the morula stage, and the expression level remained flat until the blastocyst stage. Ubc, Ppia, and Pgk1 were the three most stable reference genes in diploid and polyploid embryos.
    PLoS ONE 09/2014; 9(6):e98956. DOI:10.1371/journal.pone.0098956 · 3.23 Impact Factor
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    • "QPCR is a highly sensitive and specific method with excellent reproducibility and less post amplification procedures (Bustin et al., 2005; Valasek & Repa, 2005; Francino et al., 2006; Imène et al., 2011). That's why qPCR has become one of the favorite method for validation of microarray data or a smaller set of genes, transgenic gene expression, molecular diagnostics and biotic or abiotic stress (Arikawa et al., 2008; Kant et al., 2008; Cortleven et al., 2009; Chang et al., 2010; Di Matteo et al., 2010). It is extremely powerful technique to study the expression patterns of any gene in given conditions but a careful normalization of the data is required. "
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    ABSTRACT: 18S ribosomal RNA (18S rRNA) has been used as housekeeping gene for normalization in gene expression studies of plants. Recently, the effect of experimental conditions and nature of samples have been shown on the stability of internal control gene. Agave americana is a monocot heat tolerant plant adapted to arid conditions with Crassulacean acid metabolism (CAM). Few reports have shown the gene expression studies of this or other CAM plants due to lack of suitable reference gene. Here, we partially sequenced 18S rRNA gene of agave and evaluated its potential use as reference gene under heat stress conditions. Gene specific primers were designed from highly conserved regions of known 18S rRNA genes and amplified by using genomic DNA and transcript of Agave followed by sequencing (submitted to gene bank with accession # HM991824). To validate the potential use of Agave 18S rRNA gene for real-time PCR data normalization, we evaluated the expression stabilities of this gene in different tissues and various heat stress conditions. The plants were treated with different temperatures viz., 35ºC, 40ºC, 45ºC, 50ºC and 60ºC. The relative abundance of a heat regulated gene, Cp-sHSP (chloroplast small heat shock protein) was examined by real-time PCR. Varied levels of Cp-sHSP gene expression under different heat treatments showed the heat regulated expression. Maximum Cp-sHSP gene expression was observed in the leaves of Agave after heat stress for four hours at 45ºC. No significant difference in 18S rRNA expression was observed among control and heat treated samples. Conclusively, this 18S rRNA gene could be used as a stable internal control for normalization of real-time PCR data of A. americana. This work will help to explore many key players in the heat stress related pathways of CAM plants.
    Pakistan Journal of Botany 08/2012; 44(4):1289-1296. · 0.82 Impact Factor
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