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.

Figure 1. Specificity of primer pairs for RT-qPCR amplification.

Equal amounts of cDNAs from all tested samples were mixed as the template. 2.5% non-denaturing agarose gel electrophoresis showed amplification of a specific product of the expected size for each reference gene. M represented DNA size marker.

Figure 2. RT-qPCR CT values for the candidate reference genes.

Expression data displayed as CT values for each reference gene in all papaya samples. A line across the box is depicted as the median. The box indicates the 25th and 75th percentiles. Whiskers represent the maximum and minimum values.

Figure 3. Average expression stability values (M) of the candidate reference genes.

Average expression stability values (M) of the reference genes were measured during stepwise exclusion of the least stable reference genes. A lower M value indicated more stable expression, as analyzed by the geNorm software in papaya sample sets under different experimental conditions, including different storage temperatures (a), hot water treatment (b), modified atmosphere packaging (c), hormone treatment (d), 1-MCP fumigation treatment (e), different developmental stages (f), different tissues (g), biotic stress (h), postharvest ripening: cultivar of ‘Shuiyou 2′ (i), cultivar of ‘Hongri 1′ (j), cultivar of ‘Hongri 3′ (k). Different cultivars samples (l) and all papaya samples (m) were also given.

Figure 4. Determination of the optimal number of reference genes.

Pair-wise variation (V) calculated by geNorm to determine the minimum number of reference genes for accurate normalization in different experiment conditions. Arrow indicates the optimal number of genes for normalization in each sample sets.

Figure 5. Relative quantification of CpaEXY1 expression using validated reference genes for normalization under different experimental conditions.

(a) The validated reference gene(s) used as normalization factors were one (UBCE) or two (UBCE+CYP) most stable reference genes, and one most unstable gene (GAPDH) in postharvest ripening of ‘Shuiyou 2′ sample sets. (b) The validated reference gene(s) used as normalization factors were one (UBQ) or two (UBQ+CYP) most stable reference genes, and the most unstable one (CHY) in postharvest ripening of ‘Hongri 3′ sample sets. Reference genes validated by geNorm or NormFinder. Each value represented the means of three replicates, and vertical bars indicate the standard deviations (SD). In Figure 5b, UBQ +CYP and UBQ normalized curves belonged to left y axis, and CHY normalized curve belonged to right y axis.