Abstract
Carrot, a biennial herb of the Apiaceae family, is among the most important vegetable crops in the world. In this study, nine candidate reference genes (GAPDH, ACTIN, eIF-4α, PP2A, SAND, TIP41, UBQ, EF-1α, and TUB) were cloned from carrot. Carrot plants were subjected to abiotic stresses (heat, cold, salt, and drought) and hormone stimuli (gibberellin, salicylic acid, methyl jasmonate, and abscisic acid). The expression profiles of the candidate reference genes were evaluated in three technical and biological replicates. Real-time qPCR data analyses were performed using three commonly used Excel-based applets namely, BestKeeper, geNorm, and NormFinder. ACTIN and TUB were the most stable genes identified among all sample groups, but individual analysis revealed changes in their expression profiles. GAPDH displayed the maximum stability for most of single stresses. To further validate the suitability of the reference genes identified in this study, the expression profile of DcDREB-A1 gene (homolog of AtDREB-A1 gene of Arabidophsis) was studied in carrot. The appropriate reference genes were selected that showed stable expression under the different experimental conditions.
Highlights
Quantitative real-time reverse transcription polymerase chain reaction allows accurate high-throughput RNA quantification over a wide dynamic range at a relatively low cost; this technique has high sensitivity and has been widely used for gene expression analysis [1,2,3,4]
Accurate normalization of gene expression against an appropriate internal control is required for a valid Quantitative real-time reverse transcription polymerase chain reaction (qPCR) analysis
Leaf is a vital organ of the response to the abiotic stress and hormone signal
Summary
Quantitative real-time reverse transcription polymerase chain reaction (qPCR) allows accurate high-throughput RNA quantification over a wide dynamic range at a relatively low cost; this technique has high sensitivity and has been widely used for gene expression analysis [1,2,3,4]. Appropriate reference genes could eliminate the discrepancy that may exist in different samples and ensure the accuracy and reliability of the experimental results. Discrepancies may be due to variations in RNA expression levels and the quality and efficiency of reverse transcription. The use of reference genes to measure the temporal and spatial expressions of the target gene is widely acknowledged as a standardized method. Suitable internal controls for gene expression studies have been recognized for pepper [5], rice [6], Arabidopsis thaliana [7], Brachypodium distachyon [8], chicory [9], poplar [10], coffee [11], Oenanthe javanica (BI.)
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