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.
Highlights
Gene expression analysis is an important step to understand the roles of genes in developmental and cellular processes, such as the signaling and metabolic pathways [1]
The PCR amplification efficiency for the 21 reference genes varied from 91.1% for adenine phosphoribosyl transferase (APT) to 106.9% for TATA binding protein 2 (TBP2), and correlation coefficients ranged between 0.992 and 0.999 for APT and elongation factor 1-alpha (EF1), ubiquitin conjugating enzyme (UBCE), UBQ or Actin 2 (ACTIN), respectively (Table 2)
As no single gene has a stable expression under every experimental condition [14], it is advisable to validate the expression stability of candidate reference genes under specific experimental conditions prior to their use in RT-qPCR normalization, rather than using reference genes published elsewhere without validation [62]
Summary
Gene expression analysis is an important step to understand the roles of genes in developmental and cellular processes, such as the signaling and metabolic pathways [1]. Real-time reverse transcription PCR (RT-qPCR) has emerged as the most widely used method to quantify changes in gene expression profiles in response to different environmental conditions. It has shown important attributes such as accuracy, precision and relative ease of use due to its speed, sensitivity and specificity [2,3]. There is an urgent need to systematically evaluate the stability of potential reference genes for every particular experimental condition prior to their use in RT-qPCR normalization. Several algorithms, such as geNorm [15], NormFinder [16], BestKeeper [17], qBasePlus [18], and RefFinder [19] have been well developed to validate the most stable reference gene(s) from a panel of potential genes or candidate genes under a given set of experimental conditions [20,21]
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