Identification and evaluation of new reference genes in Gossypium hirsutumfor accurate normalization of real-time quantitative RT-PCR data

Abstract

BackgroundNormalizing through reference genes, or housekeeping genes, can make more accurate and reliable results from reverse transcription real-time quantitative polymerase chain reaction (qPCR). Recent studies have shown that no single housekeeping gene is universal for all experiments. Thus, suitable reference genes should be the first step of any qPCR analysis. Only a few studies on the identification of housekeeping gene have been carried on plants. Therefore qPCR studies on important crops such as cotton has been hampered by the lack of suitable reference genes.ResultsBy the use of two distinct algorithms, implemented by geNorm and NormFinder, we have assessed the gene expression of nine candidate reference genes in cotton: GhACT4, GhEF1α5, GhFBX6, GhPP2A1, GhMZA, GhPTB, GhGAPC2, GhβTUB3 and GhUBQ14. The candidate reference genes were evaluated in 23 experimental samples consisting of six distinct plant organs, eight stages of flower development, four stages of fruit development and in flower verticils. The expression of GhPP2A1 and GhUBQ14 genes were the most stable across all samples and also when distinct plants organs are examined. GhACT4 and GhUBQ14 present more stable expression during flower development, GhACT4 and GhFBX6 in the floral verticils and GhMZA and GhPTB during fruit development. Our analysis provided the most suitable combination of reference genes for each experimental set tested as internal control for reliable qPCR data normalization. In addition, to illustrate the use of cotton reference genes we checked the expression of two cotton MADS-box genes in distinct plant and floral organs and also during flower development.ConclusionWe have tested the expression stabilities of nine candidate genes in a set of 23 tissue samples from cotton plants divided into five different experimental sets. As a result of this evaluation, we recommend the use of GhUBQ14 and GhPP2A1 housekeeping genes as superior references for normalization of gene expression measures in different cotton plant organs; GhACT4 and GhUBQ14 for flower development, GhACT4 and GhFBX6 for the floral organs and GhMZA and GhPTB for fruit development. We also provide the primer sequences whose performance in qPCR experiments is demonstrated. These genes will enable more accurate and reliable normalization of qPCR results for gene expression studies in this important crop, the major source of natural fiber and also an important source of edible oil. The use of bona fide reference genes allowed a detailed and accurate characterization of the temporal and spatial expression pattern of two MADS-box genes in cotton.

High-quality reference genes for quantifying the transcriptional responses of Oryza sativa L. (ssp. indica and japonica) to abiotic stress conditions

“…Instead, it utilizes a mathematical modeling approach to describe the expression values of the genes and performs statistical analyses of intra-and inter-group variations in order to arrive at the stability value for a potential control gene. Because of the differences in the algorithms, the ranking of candidates in these two programs can be different [17,18,20]. Here, we used a modified version of geNorm called "geNorm PLUS " which has more effective analysis than the old version [27,33] and it allows ranking of candidate references up to the single most stable gene.…”

“…The use of EP and HNR for normalization met this criterion, and the inclusion of five genes (EP, HNR, TBC, ZCF61, and TPI) reduced the V value of the set of references substantially from 0.122 to 0.073 for our experimental conditions ( Figure S2). Normalization with multiple reference genes has been reported to give more reliable data [17,47,48]. However, the use of so many reference genes might be unduly expensive for the analysis of large numbers of experimental samples and/or conditions, and EP alone or EP with HNR would suffice.…”

Phytochrome RNAi enhances major fibre quality and agronomic traits of the cotton Gossypium hirsutum L

“…The first-strand cDNAs were diluted 1:15 with sterile water and used in expression analyses. RT-PCR reactions were carried out with intron-specific primer pairs 45 (A1341F/R; Supplementary Table S6) to check for DNA contamination, and with primers for catalytic subunit of protein phosphatase 2A gene of cotton (GhPP2A1) 46 to check the quality of the cDNAs synthesized. RT-PCR reactions were carried out using an RT-PCR kit (Sigma) according to the manufacturer's protocol.…”

Selection of optimized candidate reference genes for qRT-PCR normalization in rice (Oryza sativa L.) during Magnaporthe oryzae infection and drought

“…Among the methods commonly used to determine levels of gene expression, real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) is the method of choice, and due to its high sensitivity, specificity, and ability to provide reproducible quantification of nucleic acids, this technique has been considered a useful and available method for evaluating changes in gene expression (Bustin et al, 2005(Bustin et al, , 2009Artico et al, 2010).…”