Abstract
Quantitative reverse transcription PCR (qRT-PCR) is a sensitive technique used in gene expression studies. To achieve a reliable quantification of transcripts, appropriate reference genes are required for comparison of transcripts in different samples. However, few reference genes are available for non-model taxa, and to date, reliable reference genes in Cycas elongata have not been well characterized. In this study, 13 reference genes (ACT7, TUB, UBQ, EIF4, EF1, CLATHRIN1, PP2A, RPB2, GAPC2, TIP41, MAPK, SAMDC and CYP) were chosen from the transcriptome database of C. elongata, and these genes were evaluated in 8 different organ samples. Three software programs, NormFinder, GeNorm and BestKeeper, were used to validate the stability of the potential reference genes. Results obtained from these three programs suggested that CeGAPC2 and CeRPB2 are the most stable reference genes, while CeACT7 is the least stable one among the 13 tested genes. Further confirmation of the identified reference genes was established by the relative expression of AGAMOUSE gene of C. elongata (CeAG). While our stable reference genes generated consistent expression patterns in eight tissues, we note that our results indicate that an inappropriate reference gene might cause erroneous results. Our systematic analysis for stable reference genes of C. elongata facilitates further gene expression studies and functional analyses of this species.
Highlights
Quantitative reverse-transcription PCR has been a key technology of gene expression analysis in numerous molecular biology applications [1]
From the C. elongata transcriptome database, we retrieved thirteen putative reference genes using the sequences of A. thaliana orthologs as probes (Table 1)
Since no C. elongata relevant genetic sequences information is available online, we cloned and sequenced the reference genes according to the selected sequences
Summary
Quantitative reverse-transcription PCR (qRT-PCR) has been a key technology of gene expression analysis in numerous molecular biology applications [1]. With high throughput capability for quantification of transcript levels, qRT-PCR is considered highly sensitive, accurate, and reproducible [2]. Some variables such as the integrity, amount and purity of the RNA, as well as enzyme efficiency during cDNA synthesis and PCR amplification make it necessary to normalize the data for an accurate and reliable result [3]. A reliable reference gene in which expression is constant and stable at different developmental stages, nutritional conditions or experimental conditions is required for normalization [4]. Common reference genes or internal control genes used in qRT-PCR are housekeeping genes (HKGs) related to PLOS ONE | DOI:10.1371/journal.pone.0154384 April 28, 2016
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