Abstract
Quantitative real-time reverse transcription polymerase chain reaction (qPCR), a sensitive technique for gene expression analysis, depends on the stability of the reference genes used for data normalization. Caragana intermedia, a native desert shrub with strong drought-resistance, sand-fixing capacity and high forage value that is widespread in the desert land of west and northwest China, has not been investigated regarding the identification of reference genes suitable for the normalization of qPCR data. In this study, 10 candidate reference genes were analyzed in C. intermedia subjected to different abiotic (osmotic, salt, cold and heat) stresses, in two distinct plant organs (roots and leaves). The expression stability of these genes was assessed using geNorm, NormFinder and BestKeeper algorithms. The best-ranked reference genes differed across the different sets of samples, but UNK2, PP2A and SAND were the most stable across all tested samples. UNK2 and SAND would be appropriate for normalizing gene expression data for salt-treated roots, whereas the combination of UNK2, SAND and EF-1α would be appropriate for salt-treated leaves. UNK1, UNK2 and PP2A would be appropriate for PEG-treated (osmotic) roots, whereas the combination of TIP41 and PP2A was the most suitable for PEG-treated leaves. SAND, PP2A and TIP41 exhibited the most stable expression in heat-treated leaves. In cold-treated leaves, SAND and EF-1α were the most stably expressed. To further validate the suitability of the reference genes identified in this study, the expression levels of DREB1 and DREB2 (homologs of AtDREB1 and AtDREB2) were studied in parallel. This study is the first systematic analysis for the selection of superior reference genes for qPCR in C. intermedia under different abiotic stress conditions, and will benefit future studies on gene expression in C. intermedia and other species of the leguminous genus Caragana.
Highlights
Quantitative real-time reverse transcription polymerase chain reaction is an efficient, specific, and reproducible method for quantifying transcript expression levels, and is widely used to analyze mRNA in different organisms [1], developmental stages [2,3] and responses to abiotic and biotic stress [4,5,6,7]
The expression levels of the candidate reference genes were determined as quantification cycle (Cq) values, and the transcripts of these genes showed different levels of abundance (Figure 1)
EF-1a had the lowest Cq, indicating the highest level of expression, SAND, phosphatase 2A (PP2A), TIP41-like family protein (TIP41), TUA5, UNK1 and UNK2 were moderately expressed, F-box/kelch-repeat protein (F-box) and ACT7 were expressed at low levels
Summary
Quantitative real-time reverse transcription polymerase chain reaction (qPCR) is an efficient, specific, and reproducible method for quantifying transcript expression levels, and is widely used to analyze mRNA in different organisms [1], developmental stages [2,3] and responses to abiotic and biotic stress [4,5,6,7]. Some new reference genes were identified by microarray analyses in Arabidopsis thaliana and soybean that show highly stable expression levels [16,17]. These reference genes include SAND family protein (SAND), protein phosphatase 2A (PP2A), TIP41-like family protein (TIP41), F-box/kelch-repeat protein (F-box), phosphoenolpyruvate carboxylase-related kinase 1 (PEPKR1) and others. Systematic validation of reference genes is essential for certain experimental conditions and in different species [10] Statistical algorithms, such as geNorm [21], NormFinder [22], and BestKeeper [23], have been used to identify the best reference genes for qPCR data normalization under different experimental conditions
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
