Abstract
BackgroundStrawberry has received much attention due to its nutritional value, unique flavor, and attractive appearance. The availability of the whole genome sequence and multiple transcriptome databases allows the great possibility to explore gene functions, comprehensively. Gene expression profiles of a target gene can provide clues towards the understanding of its biological function. Quantitative real-time PCR (qRT-PCR) is a preferred method for rapid quantification of gene expression. The accuracy of the results obtained by this method requires the reference genes with consistently stable expression to normalize its data.ResultsIn present study, the expression stability of seven candidate reference genes in diverse sample subsets of different tissues and fruit developmental stages, and plant subjected to light quality and low temperature treatments was evaluated using three statistical algorithms, geNorm, NormFinder, and BestKeeper. Our data indicated that the expression stability of reference genes varied under different experimental conditions. Overall, DBP, HISTH4, ACTIN1 and GAPDH expressed much more stably. PIRUV, ACTIN2 and 18S were not recommended for normalization in given experimental conditions due to low stability. In addition, the relative expression pattern of HY5 (ELONGATED HYPOCOTYL5) was conducted to further confirm the reliability of the reference genes, which demonstrated the correct adoption of reference genes was of great importance in qRT-PCR analysis.ConclusionsExpression stability of reference genes from strawberry varied across selected experimental conditions. Systematic validation of reference genes prior to calculation of target gene expression level should be done to improve the accuracy and consistency of qRT-PCR analysis.
Highlights
Strawberry has received much attention due to its nutritional value, unique flavor, and attractive appearance
Amplification specificity and efficiency of candidate reference genes The specific primers of seven candidate reference genes were chosen for Quantitative real-time PCR (qRT-PCR), with the amplicon length ranging from 72 to 219 bp
Six primer pairs previously used as the control genes in strawberry were selected; The ACTIN1 primers were designed using Primer Premier 5.0 software and the specificity of the amplicon was confirmed by a single PCR product presence of the expected size on 2.0% agarose gel following electrophoresis (Additional file 1: Figure S1)
Summary
Strawberry has received much attention due to its nutritional value, unique flavor, and attractive appearance. Quantitative real-time PCR (qRT-PCR) is a preferred method for rapid quantification of gene expression. The accuracy of the results obtained by this method requires the reference genes with consistently stable expression to normalize its data. Gene expression analysis reflects the quantification of mRNA transcription levels of selected genes and provides a novel insight into the genes function in signaling transduction, metabolic mechanism and regulatory network. Quantitative real-time PCR (qRT-PCR) has become a very prevalent technique to determine the level of gene expression and validate the results of. An ideal reference gene should keep the expression at a constant level under various conditions and cannot be influenced by experimental parameters. Selection and validation of reference genes must be conducted in each experimental background prior to their use in qRT-PCR normalization analysis
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