Selection of reference genes for quantitative real‐time PCR in postharvest tomatoes (Lycopersicon esculentum) treated by continuous low‐voltage direct current electricity to increase secondary metabolites |
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| Authors: | Rungnapa Leelatanawit Thunyarat Saetung Sudtida Phuengwas Nitsara Karoonuthaisiri Sakamon Devahastin |
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| Affiliation: | 1. Microarray Laboratory, National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency, Khlong Luang, Pathumthani, Thailand;2. Advanced Food Processing Research Laboratory, Department of Food Engineering, Faculty of Engineering, King Mongkut's University of Technology Thonburi, Bangkok, Thailand |
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| Abstract: | Quantitative real‐time PCR (RT‐qPCR) is often used for gene expression analysis to reveal molecular mechanism of how stresses can enhance the secondary metabolites production. For RT‐qPCR to be valid, robust reference genes are required. This study validated nine candidate genes as reference genes using BestKeeper, NormFinder and geNorm methods in RT‐qPCR analysis of postharvest tomatoes subjected to electricity‐induced stress. The most stable genes as indicated by each method were EF‐1α by BestKeeper; CAC by NormFinder; and PP2Acs/TIP41 by geNorm. Due to the inconsistency in the ranking of the candidate genes by the three methods, the pairwise variation from geNorm analysis was used to calculate the minimum numbers of reference genes for an accurate normalisation and revealed that a combination of PP2Acs and TIP41 was an optimum. This reference gene combination was further validated for their stability in RT‐qPCR analysis of four carotenoid‐related genes, and more reliable expression levels were obtained. |
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| Keywords: | Electric treatment housekeeping genes internal control
Lycopersicon esculentum
quantitative real‐time PCR reference genes tomatoes |
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