Evaluation of candidate reference genes for qPCR in maize.

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.

/pdf/reference-gene-selection-for-quantitative-real-time-pcr-574fxmjnao.pdf

Reference gene selection for quantitative real-time PCR normalization in Caragana intermedia under different abiotic stress conditions.

Platycladus orientalis is a tree species that is highly resistant, widely adaptable, and long-lived, with lifespans of even thousands of years. To explore the mechanisms underlying these characteristics, gene expressions have been investigated at the transcriptome level by RNA-seq combined with a digital gene expression (DGE) technique. So, it is crucial to have a reliable set of reference genes to normalize the expressions of genes in P. orientalis under various conditions using the most accurate and sensitive method of quantitative real-time PCR (qRT-PCR). In this study, we selected 10 reference gene candidates from transcriptome data of P. orientalis, and examined their expression profiles by qRT-PCR using 29 different samples of P. orientalis, which were collected from plants of different ages, different tissues, and plants subjected to different treatments including cold, heat, salinity, polyethylene glycol (PEG), and abscisic acid (ABA). Three analytical software packages (geNorm, Bestkeeper, and NormFinder) were used to assess the stability of gene expression. The results showed that ubiquitin-conjugating enzyme E2 (UBC) and alpha-tubulin (aTUB) were the optimum pair of reference genes at all developmental stages and under all stress conditions. ACT7 was the most stable gene across different tissues and cold-treated samples, while UBQ was the most stably expressed reference gene for NaCl- and ABA-treated samples. In parallel, aTUB and UBC were used singly or in combination as reference genes to examine the expression levels of NAC (a homolog of AtNAC2) in plants subjected to various treatments with qRT-PCR. The results further proved the reliability of the two selected reference genes. Our study will benefit future research on the expression of genes in response to stress/senescence in P. orientalis and other members of the Cupressaceae.

/pdf/selection-of-reference-genes-for-quantitative-gene-2d5qtn7j51.pdf

Selection of reference genes for quantitative gene expression studies in Platycladus orientalis (Cupressaceae) Using real-time PCR.

Reliable reference selection for the accurate quantification of gene expression under various experimental conditions is a crucial step in qRT-PCR normalization. To date, only a few housekeeping genes have been identified and used as reference genes in tea plant. The validity of those reference genes are not clear since their expression stabilities have not been rigorously examined. To identify more appropriate reference genes for qRT-PCR studies on tea plant, we examined the expression stability of 11 candidate reference genes from three different sources: the orthologs of Arabidopsis traditional reference genes and stably expressed genes identified from whole-genome GeneChip studies, together with three housekeeping gene commonly used in tea plant research. We evaluated the transcript levels of these genes in 94 experimental samples. The expression stabilities of these 11 genes were ranked using four different computation programs including geNorm, Normfinder, BestKeeper, and the comparative ∆CT method. Results showed that the three commonly used housekeeping genes of CsTUBULIN1, CsACINT1 and Cs18S rRNA1 together with CsUBQ1 were the most unstable genes in all sample ranking order. However, CsPTB1, CsEF1, CsSAND1, CsCLATHRIN1 and CsUBC1 were the top five appropriate reference genes for qRT-PCR analysis in complex experimental conditions.

https://www.mdpi.com/1422-0067/15/12/22155/pdf

Identification and evaluation of reliable reference genes for quantitative real-time PCR analysis in tea plant (Camellia sinensis (L.) O. Kuntze).

https://www.uu.nl/sites/default/files/pmi_biolcontrol-alizadeh-2013.pdf

Induced systemic resistance in cucumber and Arabidopsis thaliana by the combination of Trichoderma harzianum Tr6 and Pseudomonas sp. Ps14

It has been shown that genes considered to be valid reference genes using semi-quantitative techniques (e.g. northern blot) appear to be less reliable when highly sensitive real-time PCR (qPCR) or microarrays are used. Therefore, the validation of expression stability of reference genes has become an important component of any study using such types of assay. No reference genes have been validated for expression studies of cucumber genes to date. Since the genome of this widely cultivated crop has been recently sequenced, the availability of suitable reference genes for expression analyses of the new cucumber genes is urgently required. For the purpose of normalization in studying expression of cucumber target genes, the stability of twelve reference genes in different cucumber tissues and under various stresses and growth regulators were determined in this study. These included commonly used cucumber reference genes, such as actin, EF, cyclophilin, ubiquitin and tubulin and the newly identified candidates for reference genes that encode clathrin adaptor complex subunit (CACS), F-box protein, PPA2 activator (tonoplast intrinsic protein, TIP41), mitosis protein (YSL8), protein phosphatase 2 (PDF2), helicase (HEL) and protein homolog of At4g33380. Analyses of quantitative real-time PCR data by three commonly used Excel-based applets, BestKeeper, geNorm and NormFinder, confirmed that expression stability of reference genes depends on the experimental parameters. In addition, they revealed that, except for EF, the most stable cucumber genes included mainly the new reference genes: CACS, F-box and TIP41, whereas the commonly used internal controls demonstrated various (actin, cyclophilin, ubiquitin) or much lower stability (tubulin). Hence, the authors of this study assume that the novel cucumber reference genes will enable better normalization and quantification of transcript levels in future expression studies on cucumber plants.

A. Papierniak

Papers

Identification of suitable reference genes for studying gene expression in cucumber plants subjected to abiotic stress and growth regulators