ABSTRACT
Quantitative real-time polymerase chain reaction (qRT-PCR) is a prevalent method for gene expression analysis, depending on the stability of the reference genes for data normalization. Lagerstroemia indica and L. speciosa are popular ornamental plants which are famous for the long flowering period. However, no systematic studies on reference genes in Lagerstroemia have yet been conducted. In the present study, we selected nine candidate reference genes (GAPDH, TUA, TUB, 18S, RPII, EF-1α, ATC, EIF5A and CYP) and evaluated their expression stability in different tissues during floral development of L. indica and L. speciosa using four algorithms (geNorm, NormFinder, BestKeeper and, RefFinder). Results showed that RPII and EF-1α were the most stably expressed and suitable reference genes for both of Lagerstroemia species. Moreover, ACT exhibited high expression stability in L. indica and GAPDH was a suitable reference gene for L. speciosa in different flower development stages. TUB was an unsuitable reference gene for gene expression normalization due to significant variations in expression across all samples. Finally, we verified the reliability of the selected candidate reference genes by amplifying an AGAMOUS homolog (LsAG1) of Arabidopsis thaliana. This study provides a list of suitable reference genes, thereby broadening the genetic basis of the gene expression patterns in Lagerstroemia species.
Subject(s)
Genes, Plant , Lagerstroemia/genetics , Real-Time Polymerase Chain Reaction/methods , DNA, Complementary/chemistry , DNA, Complementary/metabolism , Flowers/genetics , Gene Expression Profiling , Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)/genetics , Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)/standards , Peptide Elongation Factor 1/genetics , Peptide Elongation Factor 1/standards , RNA Polymerase II/genetics , RNA Polymerase II/standards , RNA, Plant/isolation & purification , RNA, Plant/metabolism , Real-Time Polymerase Chain Reaction/standards , Reference StandardsABSTRACT
BACKGROUND: Given the epidemic proportions of obesity worldwide and the concurrent prevalence of metabolic syndrome, there is an urgent need for better understanding the underlying mechanisms of metabolic syndrome, in particular, the gene expression differences which may participate in obesity, insulin resistance and the associated series of chronic liver conditions. Real-time PCR (qRT-PCR) is the standard method for studying changes in relative gene expression in different tissues and experimental conditions. However, variations in amount of starting material, enzymatic efficiency and presence of inhibitors can lead to quantification errors. Hence the need for accurate data normalization is vital. Among several known strategies for data normalization, the use of reference genes as an internal control is the most common approach. Recent studies have shown that both obesity and presence of insulin resistance influence an expression of commonly used reference genes in omental fat. In this study we validated candidate reference genes suitable for qRT-PCR profiling experiments using visceral adipose samples from obese and lean individuals. RESULTS: Cross-validation of expression stability of eight selected reference genes using three popular algorithms, GeNorm, NormFinder and BestKeeper found ACTB and RPII as most stable reference genes. CONCLUSIONS: We recommend ACTB and RPII as stable reference genes most suitable for gene expression studies of human visceral adipose tissue. The use of these genes as a reference pair may further enhance the robustness of qRT-PCR in this model system.
