Identification and Evaluation of Reference Genes for Accurate Transcription Normalization in Safflower under Different Experimental Conditions

Safflower (Carthamus tinctorius L.) has received a significant amount of attention as a medicinal plant and oilseed crop. Gene expression studies provide a theoretical molecular biology foundation for improving new traits and developing new cultivars. Real-time quantitative PCR (RT-qPCR) has become a crucial approach for gene expression analysis. In addition, appropriate reference genes (RGs) are essential for accurate and rapid relative quantification analysis of gene expression. In this study, fifteen candidate RGs involved in multiple metabolic pathways of plants were finally selected and validated under different experimental treatments, at different seed development stages and in different cultivars and tissues for real-time PCR experiments. These genes were ABCS, 60SRPL10, RANBP1, UBCL, MFC, UBCE2, EIF5A, COA, EF1-β, EF1, GAPDH, ATPS, MBF1, GTPB and GST. The suitability evaluation was executed by the geNorm and NormFinder programs. Overall, EF1, UBCE2, EIF5A, ATPS and 60SRPL10 were the most stable genes, and MBF1, as well as MFC, were the most unstable genes by geNorm and NormFinder software in all experimental samples. To verify the validation of RGs selected by the two programs, the expression analysis of 7 CtFAD2 genes in safflower seeds at different developmental stages under cold stress was executed using different RGs in RT-qPCR experiments for normalization. The results showed similar expression patterns when the most stable RGs selected by geNorm or NormFinder software were used. However, the differences were detected using the most unstable reference genes. The most stable combination of genes selected in this study will help to achieve more accurate and reliable results in a wide variety of samples in safflower.     

Evaluation and Validation of Housekeeping Genes as Reference for Gene Expression Studies in Pigeonpea (Cajanus cajan) Under Drought Stress Conditions

Gene expression analysis using quantitative real-time PCR (qRT-PCR) is a very sensitive technique and its sensitivity depends on the stable performance of reference gene(s) used in the study. A number of housekeeping genes have been used in various expression studies in many crops however, their expression were found to be inconsistent under different stress conditions. As a result, species specific housekeeping genes have been recommended for different expression studies in several crop species. However, such specific housekeeping genes have not been reported in the case of pigeonpea (Cajanus cajan) despite the fact that genome sequence has become available for the crop. To identify the stable housekeeping genes in pigeonpea for expression analysis under drought stress conditions, the relative expression variations of 10 commonly used housekeeping genes (EF1α, UBQ10, GAPDH, 18SrRNA, 25SrRNA, TUB6, ACT1, IF4α, UBC and HSP90) were studied on root, stem and leaves tissues of Asha (ICPL 87119). Three statistical algorithms geNorm, NormFinder and BestKeeper were used to define the stability of candidate genes. geNorm analysis identified IF4α and TUB6 as the most stable housekeeping genes however, NormFinder analysis determined IF4α and HSP90 as the most stable housekeeping genes under drought stress conditions. Subsequently validation of the identified candidate genes was undertaken in qRT-PCR based gene expression analysis of uspA gene which plays an important role for drought stress conditions in pigeonpea. The relative quantification of the uspA gene varied according to the internal controls (stable and least stable genes), thus highlighting the importance of the choice of as well as validation of internal controls in such experiments. The identified stable and validated housekeeping genes will facilitate gene expression studies in pigeonpea especially under drought stress conditions.     

Identification and Validation of Reference Genes for Quantitative Real-Time PCR in Drosophila suzukii (Diptera: Drosophilidae)

To accurately evaluate gene expression levels and obtain more accurate quantitative real-time RT-PCR (qRT-PCR) data, normalization relative to reliable reference gene(s) is required. Drosophila suzukii, is an invasive fruit pest native to East Asia, and recently invaded Europe and North America, the stability of its reference genes have not been previously investigated. In this study, ten candidate reference genes (RPL18, RPS3, AK, EF-1β, TBP, NADH, HSP22, GAPDH, Actin, α-Tubulin), were evaluated for their suitability as normalization genes under different biotic (developmental stage, tissue and population), and abiotic (photoperiod, temperature) conditions. The three statistical approaches (geNorm, NormFinder and BestKeeper) and one web-based comprehensive tool (RefFinder) were used to normalize analysis of the ten candidate reference genes identified α-Tubulin, TBP and AK as the most stable candidates, while HSP22 and Actin showed the lowest expression stability. We used three most stable genes (α-Tubulin, TBP and AK) and one unstably expressed gene to analyze the expression of P-glycoprotein in abamectin-resistant and sensitive strains, and the results were similar to reference genes α-Tubulin, TBP and AK, which show good stability, while the result of HSP22 has a certain bias. The three validated reference genes can be widely used for quantification of target gene expression with qRT-PCR technology in D.suzukii.     

Selection and Evaluation of Reference Genes for Expression Analysis Using qRT-PCR in the Beet Armyworm Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae)

Quantitative real-time PCR (qRT-PCR) is a reliable and reproducible technique for measuring and evaluating changes in gene expression. The most common method for analyzing qRT-PCR data is to normalize mRNA levels of target genes to internal reference genes. Evaluating and selecting stable reference genes on a case-by-case basis is critical. The present study aimed to facilitate gene expression studies by identifying the most suitable reference genes for normalization of mRNA expression in qRT-PCR analysis of the beet armyworm Spodoptera exigua (Lepidoptera: Noctuidae). For this purpose, three software tools (geNorm, NormFinder and BestKeeper) were used to investigate 10 candidate reference genes in nine developmental stages and five different tissues (epidermis, head, midgut, fat body and hemolymph) in three larval physiological stages (molting, feeding and wandering stages) of, S. exigua. With the exception of 18S ribosomal RNA (18S), all other candidate genes evaluated, β-actin1(ACT1), β-actin2 (ACT2), elongation factor1(EF1), elongation factor 2 (EF2), Glyceralde hyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein L10 (L10), ribosomal protein L17A (L17A), superoxide dismutase (SOD), α-tubulin (TUB),proved to be acceptable reference genes. However, their suitability partly differed between physiological stages and different tissues. L10, EF2 and L17A ranked highest in all tissue sample sets. SOD, ACT2, GAPDH, EF1 and ACT1 were stably expressed in all developmental stage sample sets; ACT2, ACT1 and L10 for larvae sample sets; GAPDH, ACT1 and ACT2 for pupae and adults; SOD and L17A for males; and EF2 and SOD for females. The expression stability of genes varied in different conditions. The findings provided here demonstrated, with a few exceptions, the suitability of most of the 10 reference genes tested in tissues and life developmental stages. Overall, this study emphasizes the importance of validating reference genes for qRT-PCR analysis in S. exigua.     

ACTB,CDKN1B,GAPDH, GRB2, RHOA and SDCBP Were Identified as Reference Genes in Neuroendocrine Lung Cancer via the nCounter Technology

BackgroundNeuroendocrine lung cancer (NELC) represents 25% of all lung cancer cases and large patient collectives exist as formalin-fixed, paraffin-embedded (FFPE) tissue only. FFPE is controversially discussed as source for molecular biological analyses and reference genes for NELC are poorly establishes.ResultsRNA integrity (RIN) ranged from 1.8 to 2.6 and concentrations from 34 to 2,109 ng/μl. However, the nCounter technology gave evaluable results for all samples tested. ACTB, CDKN1B, GAPDH, GRB2, RHOA and SDCBP were identified as constantly expressed genes with high stability (M-)values according to geNorm, NormFinder and coefficients of correlation.ConclusionFFPE-derived mRNA is suitable for molecular biological investigations via the nCounter technology, although it is highly degraded. ACTB, CDKN1B, GAPDH, GRB2, RHOA and SDCBP are potent reference genes in neuroendocrine tumors of the lung.     

Reference gene selection for real-time quantitative polymerase chain reaction normalization in “Swingle” citrumelo under drought stress

We describe the first systematic evaluation of reference genes for use in real-time quantitative polymerase chain reaction (qPCR) for water deficit stress studies in the citrus rootstock “Swingle” citrumelo. The expression levels of seven reference genes—cyclophilin (CYP), cathepsin (CtP), actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), elongation factor 1α (EF1α), β-tubulin (TUB), and ADP ribosylation factor (ADP)—during drought stress were tested using geNorm and NormFinder programs. Results from four experimental conditions indicated that EF1α and ADP were the most stable reference genes. Relative expression levels of Δ1-pyrroline-5-carboxylate synthetase (P5CS) was used for reference gene validation.     

Selection of reference genes for Harmonia axyridis (Coleoptera: Coccinellidae) feeding on different diets

The selection of a stable reference gene is vital to gene expression studies and to improving the accuracy of RT-qPCR data. With the deep research on the artificial feed of the Harmonia axyridis, there is an increasing need to evaluate the effects of feed on insects at the molecular level. To identify a reference gene to assess the expression of related genes in Harmonia axyridis (Pallas), ensure the reliability of target gene expression analysis using real-time PCR. Especially for H. axyridis were fed Rhopalosiphum padi (L.) or an artificial diet. In this study, the expression profiles of nine candidate reference genes, including 28SrRNA, 18SrRNA, RPS23, EF1, Actin, ATPase, GAPDH, UBI, RPL13 from different H. axyridis tissues (head, thorax, wing, leg, ovary, and fat body) were investigated. The stability of the nine candidate genes was assessed using geNorm, NormFinder, BestKeeper and RefFinder software, and a comprehensive analysis showed that EF1 is a suitable reference gene for eating different diets of different organizations from H. axyridis. 28SrRNA, 18SrRNA, and RPS23 can also be used as reference genes, but Actin, ATPase, RPL13 are not suitable as an internal reference gene.     

Expression Profiling in Bemisia tabaci under Insecticide Treatment: Indicating the Necessity for Custom Reference Gene Selection

Finding a suitable reference gene is the key for qRT-PCR analysis. However, none of the reference gene discovered thus far can be utilized universally under various biotic and abiotic experimental conditions. In this study, we further examine the stability of candidate reference genes under a single abiotic factor, insecticide treatment. After being exposed to eight commercially available insecticides, which belong to five different classes, the expression profiles of eight housekeeping genes in the sweetpotato whitefly, Bemisia tabaci, one of the most invasive and destructive pests in the world, were investigated using qRT-PCR analysis. In summary, elongation factor 1α (EF1α), α-tubulin (TUB1α) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were identified as the most stable reference genes under the insecticide treatment. The initial assessment of candidate reference genes was further validated with the expression of two target genes, a P450 (Cyp6cm1) and a glutathione S-transferase (GST). However, ranking of reference genes varied substantially among intra- and inter-classes of insecticides. These combined data strongly suggested the necessity of conducting custom reference gene selection designed for each and every experimental condition, even when examining the same abiotic or biotic factor.     

Validation of reference genes for gene expression analysis in Valsa mali var. mali using real-time quantitative PCR

Valsa mali var. mali (Vmm), is the predominant species of apple valsa canker in China. Modern analysis of genes involved in virulence or pathogenicity usually implicate gene expression analysis most often performed using real-time quantitative polymerase chain reaction (RT-qPCR). However, for relative gene expression analysis pertinent reference genes have to be validated before using them as internal reference. This has not been reported for Vmm, so far. Therefore, eight commonly used housekeeping genes (ACT, CYP, EF1-α, G6PDH, GAPDH, L13, TUB, and UBQ) were cloned and evaluated for their expression stability by geNorm and NormFinder. Overall, all of the candidate reference genes were found to be suitable for gene expression analysis. After analysis of 10 samples from different strains and abiotic stress treatments, G6PDH appeared to be the most suitable reference gene, whereas GAPDH was the least suitable. Moreover, taking G6PDH combined with L13 or CYP as reference genes, improved the reliability of RT-qPCR significantly. The influence of the reference system on expression data was demonstrated by analyzing Vmmpg-1 encoding an endo-polygalacturonase gene. Pectinases are considered key pathogenicity factors for this fungus. In order to better understand the role of pectinases in pathogenicity of Vmm, RT-qPCR was used for expression analysis. Our results may provide a guideline for future studies on gene expression of V. mali var. mali by using RT-qPCR.     

Reference Gene Selection for Quantitative Real-Time PCR Normalization in Reaumuria soongorica

Despite its superiority for evaluating gene expression, real-time quantitative polymerase chain reaction (qPCR) results can be significantly biased by the use of inappropriate reference genes under different experimental conditions. Reaumuria soongorica is a dominant species of desert ecosystems in arid central Asia. Given the increasing interest in ecological engineering and potential genetic resources for arid agronomy, it is important to analyze gene function. However, systematic evaluation of stable reference genes should be performed prior to such analyses. In this study, the stabilities of 10 candidate reference genes were analyzed under 4 kinds of abiotic stresses (drought, salt, dark, and heat) within 4 accessions (HG010, HG020, XGG030, and XGG040) from 2 different habitats using 3 algorithms (geNorm, NormFinder, and BestKeeper). After validation of the ribulose-1,5-bisphosphate carboxylase/oxygenase large unite (rbcL) expression pattern, our data suggested that histone H2A (H2A) and eukaryotic initiation factor 4A-2 (EIF4A2) were the most stable reference genes, cyclophilin (CYCL) was moderate, and elongation factor 1α (EF1α) was the worst choice. This first systematic analysis for stably expressed genes will facilitate future functional analyses and deep mining of genetic resources in R. soongorica and other species of the Reaumuria genus.     

Reference gene selection for transcriptional profiling by RT-qPCR in the 28-spotted larger potato ladybird

Henosepilachna vigintioctomaculata is one of the most serious defoliates attacking potatoes. However, studies on functional genes have greatly been limited due to the insufficiency of effective and stable endogenous references to normalize RT-qPCR data. In this report, nine housekeeping genes (RPL4, RPL6, RPL13, RPL32, RPS18, ACT, EF1α, GAPDH and α-TUB) involved in different biological processes were selected. Their expression levels under diverse experimental conditions including developmental stages, tissues, temperatures and host plants were determined using RT-qPCR technology. The tested candidate genes were comprehensively ranked based on five alternative stability analysis methods (Ct value, geNorm, NormFinder, BestKeeper and ReFinder). The results revealed that the optimal internal reference genes varied under different experimental conditions. Any gene pair among the five candidates (RPL4, RPL13, RPL32, RPS18 and EF1α) was a suitable reference gene set under different temperatures and on different host plants. A combination of RPL6 and RPL13 was recommended as the best reference gene set across different developmental stages. A pair of RPS18 and EF1α was ranked as the optimal reference gene combination within different tissues. The most suitable reference genes were RPS18 and RPL13 under four different experimental conditions. Our findings not only establish an accurate and reliable normalization of RT-qPCR data, but also lay a solid foundation for further functional gene researches in H. vigintioctomaculata.