Evaluation of internal control genes for quantitative realtime PCR analyses for studying fruit development of dwarf tomato cultivar ‘Micro-Tom’

Abstract

Quantitative real-time PCR (qRT-PCR) is widely used to analyze the expression profiles of the genes of interest. In order to obtain accurate quantification data, normalization by using reliable internal control genes is essential. In this study, we evaluated the stability and applicability of eight internal control gene candidates for analyzing gene expression during fruit development in dwarf tomato cultivar Micro-Tom. We collected seventeen different samples from flowers and fruits at different developmental stages, and estimated the expression stability of the candidate genes by two statistical algorithms, geNorm and NormFinder. The combined ranking order and qRT-PCR analyses for expression profiles of SlYABBY2a, SlYABBY1a, FRUITFULL1 and APETALA2c suggested that EXPRESSED was the most stable and reliable internal control gene among the candidates. Our analysis also suggested that RPL8 was also suitable if the sample group is limited to fruits at different maturation stages. In addition to EXPRESSED, GAPDH was also applicable for relative quantitation to monitor gene expression profiles through fruit development from pistil to pericarp.

Stimulation of Tomato Drought Tolerance by PHYTOCHROME A and B1B2 Mutations

“…The ΔC T of WT under control conditions was used as a subtrahend factor in the ΔΔC T subtraction formula for comparison with phy mutants under control and drought stress conditions, as in the following: ∆∆CT (∆CT (Tested) − ∆CT, (WT under control) ). The tomato-expressed sequence gene ( EXPRESSED ) (Gene ID: Solyc07g025390.2.1) was used as a reference endogenous control for gene expression analyses [ 76 ].…”

“…The following supporting information can be downloaded at: . References [ 76 , 77 , 78 ] are cited in the supplementary materials.…”

The selection and validation of reference genes for quantitative real-time PCR studies in near-isogenic susceptible and resistant tomato lines, infected with the geminivirus tomato curly stunt virus

“…The first step of real-time PCR experiments for gene expression studies requires the selection and validation of reference genes for data normalization [ 19 , 30 ]. There are many reports on validation of candidate reference genes in tomato in response to various stimuli [ 23 , 24 , 31 – 36 ]. In all these studies carried out in tomato plants, fruits and seeds, the most suitable reference genes that were identified varied and no two genes were found to be stably expressed across all experimental conditions.…”

Genome‐wide identification and expression analysis of Na+/H+antiporter (NHX) genes in tomato under salt stress

“…Gene‐specific forward and reverse primers for members of SlNHX gene family were designed using MacVector v18.2 software (MacVector Inc., Cary, NC, USA) (Table 1 ). The EXPRESSED gene was used as housekeeping for normalization in RT‐qPCR analyses (Choi et al, 2018 ). The RT‐qPCR conditions: initial denaturation at 95°C for 8 min, 40 cycles of 95°C denaturation for 15 s, 55°C annealing for 30 s, and 72°C extension for 30 s. Three biological replicates were used for each sample.…”