Evaluation of New Reference Genes in Papaya for Accurate Transcript Normalization under Different Experimental Conditions

Abstract

Real-time reverse transcription PCR (RT-qPCR) is a preferred method for rapid and accurate quantification of gene expression studies. Appropriate application of RT-qPCR requires accurate normalization though the use of reference genes. As no single reference gene is universally suitable for all experiments, thus reference gene(s) validation under different experimental conditions is crucial for RT-qPCR analysis. To date, only a few studies on reference genes have been done in other plants but none in papaya. In the present work, we selected 21 candidate reference genes, and evaluated their expression stability in 246 papaya fruit samples using three algorithms, geNorm, NormFinder and RefFinder. The samples consisted of 13 sets collected under different experimental conditions, including various tissues, different storage temperatures, different cultivars, developmental stages, postharvest ripening, modified atmosphere packaging, 1-methylcyclopropene (1-MCP) treatment, hot water treatment, biotic stress and hormone treatment. Our results demonstrated that expression stability varied greatly between reference genes and that different suitable reference gene(s) or combination of reference genes for normalization should be validated according to the experimental conditions. In general, the internal reference genes EIF (Eukaryotic initiation factor 4A), TBP1 (TATA binding protein 1) and TBP2 (TATA binding protein 2) genes had a good performance under most experimental conditions, whereas the most widely present used reference genes, ACTIN (Actin 2), 18S rRNA (18S ribosomal RNA) and GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) were not suitable in many experimental conditions. In addition, two commonly used programs, geNorm and Normfinder, were proved sufficient for the validation. This work provides the first systematic analysis for the selection of superior reference genes for accurate transcript normalization in papaya under different experimental conditions.

Reference gene selection for gene expression studies in lily using quantitative real-time PCR

“…For Lilium formosanum, Volvox carteri, and Solanum melongena, 18S displays the most stable expression among samples both from different developmental stages and different stress treatments. However, in our study, 18S performed poorly, which was similar to the results obtained in papaya (Zhu et al, 2012).…”

Selection of reliable reference genes for RT-qPCR studies in Octopus vulgaris paralarvae during development and immune-stimulation

“…Results from the three approaches were subsequently analyzed according to Zhu et al (2012). This method gives a comprehensive ranking with the overall results obtained by the three algorithms used, assigning a number to each gene (between 1 to the most stable to 6 to the least stable one) according to their position …”

Selection of reference genes for quantitative real-time PCR normalization in creeping bentgrass involved in four abiotic stresses

“…To our knowledge, the current study is the first to identify stable reference genes in leaves and roots of creeping bentgrass under different abiotic stresses (salt, drought, heat, and cold) using the combination of GeNorm, NormFinder, BestKeeper, and RefFinder (Andersen et al 2004;Vandesompele et al 2002). The previous study indicated that different reference genes could be determined by three different softwares (GeNorm, NormFinder, and BestKeeper) based on different mathematical methods or parameters (Chen et al 2014;Huang et al 2014;Zhu et al 2012). Therefore, a comprehensive analysis method named RefFinder was used to achieve reliable results based on the data of different softwares.…”

“…The comprehensive rankings of candidate references shown in Table 2 were determined by RefFinder tool (http://www.leonxie.com/referencegene.php) based on different software programs including GeNorm, NormFinder, and BestKeeper (Chen et al 2014;Huang et al 2014;Zhu et al 2012). PP2A, CACS, and GAPDH were identified as the three most stable reference genes in the total samples.…”