Expressed Reference Genes Research Articles

Reference gene identification for gene expression analysis in rice under different metal stress

Real-time quantitative polymerase chain reaction (RT-qPCR) is the most common approach to quantify changes in gene expression. Appropriate internal reference genes are essential for normalization of data of RT-qPCR. In the present study, we identified suitable reference genes for analysis of gene expression in rice seedlings subjected to different heavy metal stresses such as deficiencies of iron and zinc and toxicities of cobalt, cadmium and nickel. First, from publically available RNA-Seq data we identified 10 candidate genes having stable expression. We also included commonly used house-keeping gene OsUBQ5 (Ubiquitin 5) in our analysis. Expression stability of all the 11 genes was determined by two independent tools, NormFinder and geNorm. Our results show that selected candidate reference genes have higher stability in their expression compared to that of OsUBQ5. Genes with locus ID LOC_Os03g16690, encoding an oxysterol-binding protein (OsOBP) and LOC_Os01g56580, encoding Casein Kinase_1a.3 (OsCK1a.3) were identified to be the most stably expressed reference genes under most of the conditions tested. Finally, the study reveals that it is better to use a specific reference gene for a specific heavy metal stress condition rather than using a common reference gene for multiple heavy metal stress conditions. The reference genes identified here would be very useful for gene expression studies under heavy metal stresses in rice.

Validation of Reference Genes via qRT-PCR in Multiple Conditions in Brandt's Voles, Lasiopodomys brandtii.

Simple SummaryThis study validated the stability of the expression profiles of nine common candidate reference genes (Gapdh, Hprt1, β-actin, PPIA, Rpl13a, Tbp, Sdha, Hmbs, and B2M) using qRT-PCR in different tissues, developmental stages, and photoperiods. None of these genes were suitable as optimal reference genes at 4 weeks postnatal in different tissues. Under different developmental stages in the hypothalamus, B2M for males and Rpl13a for females were suitable as reference genes. Under different photoperiods in the hypothalamus, none of the selected genes were suitable as reference genes at 6 weeks postnatal, β-actin and PPIA were the optimal reference genes at 12 weeks postnatal, while Hprt1, β-actin, PPIA, Hmbs, and B2M were excellent reference genes at 24 weeks postnatal.The choice of optimal reference gene is challenging owing to the varied expression of reference genes in different organs, development stages, and experimental treatments. Brandt’s vole (Lasiopodomys brandtii) is an ideal animal to explore the regulatory mechanism of seasonal breeding, and many studies on this vole involve gene expression analysis using quantitative real-time polymerase chain reaction (qRT-PCR). In this study, we used the method of the coefficient of variation and the NormFinder algorithm to evaluate the performance of nine commonly used reference genes Gapdh, Hprt1, β-actin, PPIA, Rpl13a, Tbp, Sdha, Hmbs, and B2M using qRT-PCR in eight different tissues, five developmental stages, and three different photoperiods. We found that all nine genes were not uniformly expressed among different tissues. B2M and Rpl13a were the optimal reference genes for different postnatal development stages in the hypothalamus for males and females, respectively. Under different photoperiods in the hypothalamus, none of the selected genes were suitable as reference genes at 6 weeks postnatal; β-actin and PPIA were the optimal reference genes at 12 weeks postnatal; Hprt1, β-actin, PPIA, Hmbs, and B2M were excellent reference genes at 24 weeks postnatal. The present study provides a useful basis for selecting the appropriate reference gene in Lasiopodomys brandtii.

Spatio-temporal selection of reference genes in the two congeneric species of Glycyrrhiza

Glycyrrhiza, a genus of perennial medicinal herbs, has been traditionally used to treat human diseases, including respiratory disorders. Functional analysis of genes involved in the synthesis, accumulation, and degradation of bioactive compounds in these medicinal plants requires accurate measurement of their expression profiles. Reverse transcription quantitative real-time PCR (RT-qPCR) is a primary tool, which requires stably expressed reference genes to serve as the internal references to normalize the target gene expression. In this study, the stability of 14 candidate reference genes from the two congeneric species G. uralensis and G. inflata, including ACT, CAC, CYP, DNAJ, DREB, EF1, RAN, TIF1, TUB, UBC2, ABCC2, COPS3, CS, R3HDM2, were evaluated across different tissues and throughout various developmental stages. More importantly, we investigated the impact of interactions between tissue and developmental stage on the performance of candidate reference genes. Four algorithms, including geNorm, NormFinder, BestKeeper, and Delta Ct, were used to analyze the expression stability and RefFinder, a comprehensive software, provided the final recommendation. Based on previous research and our preliminary data, we hypothesized that internal references for spatio-temporal gene expression are different from the reference genes suited for individual factors. In G. uralensis, the top three most stable reference genes across different tissues were R3HDM2, CAC and TUB, while CAC, CYP and ABCC2 were most suited for different developmental stages. CAC is the only candidate recommended for both biotic factors, which is reflected in the stability ranking for the spatio (tissue)-temporal (developmental stage) interactions (CAC, R3HDM2 and DNAJ). Similarly, in G. inflata, COPS3, R3HDM2 and DREB were selected for tissues, while RAN, COPS3 and CS were recommended for developmental stages. For the tissue-developmental stage interactions, COPS3, DREB and ABCC2 were the most suited reference genes. In both species, only one of the top three candidates was shared between the individual factors and their interactions, specifically, CAC in G. uralensis and COPS3 in G. inflata, which supports our overarching hypothesis. In summary, spatio-temporal selection of reference genes not only lays the foundation for functional genomics research in Glycyrrhiza, but also facilitates these traditional medicinal herbs to reach/maximize their pharmaceutical potential.

Reference gene selection for qRT-PCR analyses of luffa (Luffa cylindrica) plants under abiotic stress conditions

Selecting suitable internal reference genes is an important prerequisite for the application of quantitative real-time PCR (qRT-PCR). However, no systematic studies have been conducted on reference genes in luffa. In this study, seven reference genes were selected, and their expression levels in luffa plants exposed to various simulated abiotic stresses [i.e., cold, drought, heat, salt, H2O2, and abscisic acid (ABA) treatments] were analyzed by qRT-PCR. The stability of the reference gene expression levels was validated using the geNorm, NormFinder, BestKeeper, and RefFinder algorithms. The results indicated that EF-1α was the most stably expressed and suitable reference gene overall and for the heat, cold, and ABA treatments. Additionally, UBQ expression was stable following the salt treatment, whereas TUB was identified as a suitable reference gene for H2O2 and drought treatments. The reliability of the selected reference genes was verified by analyzing the expression of copper/zinc superoxide dismutase (Cu/Zn-SOD) gene in luffa. When the most unstable reference genes were used for data normalizations, the resulting expression patterns had obvious biases when compared with the expression patterns for the most ideal reference genes used alone or combined. These results will be conducive to more accurate quantification of gene expression levels in luffa.

Candidate reference genes for quantitative gene expression analysis in Lagerstroemia indica.

Quantitative gene expression analysis by qPCR requires reference genes for normalization. Lagerstroemia indica (crape myrtle) is a popular ornamental plant in the world, but suitable endogenous reference genes are lacking. To find suitable reference genes, we evaluated the stabilities of nine candidate genes in six experimental data sets: six different tissues, three leaf colors, nine flower colors, and under three abiotic stresses (salt, drought, cold) using four statistical algorithms. A target gene LiMYB56 (homolog of Arabidopsis MYB56) was used to verify the authenticity and accuracy of the candidate reference genes. The results showed that the combination of two stably expressed reference genes, rather than a single reference gene, improved the accuracy of the qPCR. LiEF1α-2 + LiEF1α-3 was best for the tissue, salt treatment, and drought treatment sets; LiEF1α-2 + LiEF1α-1 was optimal for leaf color; LiEF1α-2 + LiACT7 was optimal for cold treatment; and LiUBC + LiEF1α-1 was best for the flower color set. Notably, LiEF1α-2 had high expression stability in all six experimental sets, implying it may be a good reference gene for expression studies in L. indica. Our results will facilitate future gene expression studies in L. indica.

Gene Expression Imputation Across Multiple Tissue Types Provides Insight Into the Genetic Architecture of Frontotemporal Dementia and Its Clinical Subtypes

BackgroundThe etiology of frontotemporal dementia (FTD) is poorly understood. To identify genes with predicted expression levels associated with FTD, we integrated summary statistics with external reference gene expression data using a transcriptome-wide association study approach. MethodsFUSION software was used to leverage FTD summary statistics (all FTD: n = 2154 cases, n = 4308 controls; behavioral variant FTD: n = 1337 cases, n = 2754 controls; semantic dementia: n = 308 cases, n = 616 controls; progressive nonfluent aphasia: n = 269 cases, n = 538 controls; FTD with motor neuron disease: n = 200 cases, n = 400 controls) from the International FTD-Genomics Consortium with 53 expression quantitative loci tissue type panels (n = 12,205; 5 consortia). Significance was assessed using a 5% false discovery rate threshold. ResultsWe identified 73 significant gene–tissue associations for FTD, representing 44 unique genes in 34 tissue types. Most significant findings were derived from dorsolateral prefrontal cortex splicing data (n = 19 genes, 26%). The 17q21.31 inversion locus contained 23 significant associations, representing 6 unique genes. Other top hits included SEC22B (a gene involved in vesicle trafficking), TRGV5, and ZNF302. A single gene finding (RAB38) was observed for behavioral variant FTD. For other clinical subtypes, no significant associations were observed. ConclusionsWe identified novel candidate genes (e.g., SEC22B) and previously reported risk regions (e.g., 17q21.31) for FTD. Most significant associations were observed in dorsolateral prefrontal cortex splicing data despite the modest sample size of this reference panel. This suggests that our findings are specific to FTD and are likely to be biologically relevant highlights of genes at different FTD risk loci that are contributing to the disease pathology.

Variation in Expression of Reference Genes across Life Stages of a Bee, Megachile rotundata.

Simple SummaryReference genes are key to normalizing expression data across samples of organisms collected after different treatments are applied, but often, reference genes are not properly validated for this purpose. In this report, we screened several genes for a solitary bee, Megachile rotundata, and identified two (RPS18, and RPL8) with very stable expression levels across all life stages of the bee, and under a variety of environmental conditions, including during and after diapause. These genes should make good reference genes. We also identified other genes with stable expression, even if used only for a limited number of developmental stages. This information is important for future gene expression studies on these bees, but it also demonstrates the importance of validating reference genes in general.The alfalfa leafcutting bee, Megachile rotundata is widely used in the western United States as a pollinator for alfalfa seed production. Unfortunately, immatures experience high mortality in agriculturally managed populations. Quantified gene expression could be used to identify how this bee responds during different life stages to pathogens, environmental toxins, and other stresses, but stably expressed reference genes are needed to normalize transcription data. We evaluated twelve candidate genes for their transcription stability across different life stages, including during and after diapause. RPS18 and RPL8 were the two most stably expressed genes, followed by RPS5 and RPL27A. These genes were also very stable even during and after diapause, while the most variable genes being APN, PMIIM, NPC2, and Cr-PII had increased expression levels during larval growth and were also variable during and after diapause. The four reference genes we identified in M. rotundata may prove useful for transcriptomic studies in other bees as well, such as honey bees.

Selection of Suitable Reference Genes for qRT-PCR Gene Expression Studies in Rice.

With a widely established use of quantitative real-time PCR (qRT-PCR) for gene expression analysis, reliable and stable expression of reference genes is often discussed. Suitable reference genes should show less variation of expression across the target samples and allow for error minimization by normalization of qRT-PCR data. Therefore, selection of reliable reference genes is essential for accurate results and to support the conclusions drawn on expression levels of genes under study. In this chapter, we describe the workflow for selection and evaluation of reference genes in rice, including identification of candidate genes by using Genevestigator® and evaluation of expression stability using various algorithms. The ranking of the genes guides qRT-PCR performance and data analysis. This protocol used rice as an example but is not limited to rice, and could be applied to other species as well.

Quality Evaluation of Reference Gene Expression on Different Tissues in Adults of Tropical Gar Atractosteus tropicus

Tropical gar (Atractosteus tropicus) is an ancestral subtropical fish species in southeastern Mexico, which has great potential as a model species for physiological, biomedical and genomic studies. The quantification of gene expression through RT-qPCR is one of the most commonly used techniques, due to its precision, sensitivity and high performance, particularly in gene expression to compare between cells, tissues and organs; as well as different populations, stages of development, metabolism, among other conditions. This study analyzed the stability and normalization of six commonly used reference genes such as alpha elongation factor (ef1-α), beta-actin (actb), 18S ribosomal RNA (18s rrna), beta-2-microglobulin (b2m), tubulin alpha (α-tub) and glyceraldehyde 3-phosphate dehydrogenase (gapdh) in the different tissues of the intestine, muscle, gill, stomach, brain and liver in adult males of A. tropicus from reared in captivity through three BestKeeper, geNorm and NormFinder algorithms. Based on our results we can conclude that in the three BestKeeper, NormFinder and geNorm algorithms, the most stable genes are ef1, followed by 18s rrna and actb where the gene stability will depend on specific tissue to analyze in tropical gar A. tropicus adults.

Selection and validation of reference genes for qPCR in the human dermatophyte Trichophyton rubrum exposed to different carbon sources which promote adhesion-inducing conditions.

The present study aimed to identify reference genes for qPCR analysis of T.rubrum growth in culture media which promote adhesion-inducing conditions to the host tissue. We investigated the suitability of six candidate reference genes: β-act, β-tub, ef1-α, gapdh, sdha and rpl2 in reference strain of Trichophyton rubrum in response to different environmental stimuli. The stability of these genes was determined by NormFinder, geNorm and BestKeeper software. Our data obtained from the three algorithms revealed that mRNA expression levels of two candidate reference genes, ef1-α and β-tub, remained the most stable in response to different carbon sources, while different sample sets had their own most stable reference genes, highlighting the importance of the choice of internal controls in qPCR experiments. We then checked the stability of ef1-α and β-tub reference genes expression in different T.rubrum strains, suggesting that these two genes are reliable for normalisation of qPCR. Finally, we validated the suitability of selected reference genes as internal controls for target gene (SUB3) using the 2-ΔΔCt method. The best result indicating an increase of SUB3 transcript of T.rubrum was found when the two the most stable reference (ef1-α and β-tub ) genes were used, as revealed by all three algorithms. We recommend the use of ef1-α and β-tub as reference genes for qPCR analysis of target gene expression in T.rubrum exposed to different carbon sources which promote adhesion-inducing conditions.

Expression of Putative Defense Responses in Cannabis Primed by Pseudomonas and/or Bacillus Strains and Infected by Botrytis cinerea.

Cannabis (Cannabis sativa L.) offers many industrial, agricultural, and medicinal applications, but is commonly threatened by the gray mold disease caused by the fungus Botrytis cinerea. With few effective control measures currently available, the use of beneficial rhizobacteria represents a promising biocontrol avenue for cannabis. To counter disease development, plants rely on a complex network of inducible defense pathways, allowing them to respond locally and systemically to pathogens attacks. In this study, we present the first attempt to control gray mold in cannabis using beneficial rhizobacteria, and the first investigation of cannabis defense responses at the molecular level. Four promising Pseudomonas (LBUM223 and WCS417r) and Bacillus strains (LBUM279 and LBUM979) were applied as single or combined root treatments to cannabis seedlings, which were subsequently infected by B. cinerea. Symptoms were recorded and the expression of eight putative defense genes was monitored in leaves by reverse transcription quantitative polymerase chain reaction. The rhizobacteria did not significantly control gray mold and all infected leaves were necrotic after a week, regardless of the treatment. Similarly, no systemic activation of putative cannabis defense genes was reported, neither triggered by the pathogen nor by the rhizobacteria. However, this work identified five putative defense genes (ERF1, HEL, PAL, PR1, and PR2) that were strongly and sustainably induced locally at B. cinerea’s infection sites, as well as two stably expressed reference genes (TIP41 and APT1) in cannabis. These markers will be useful in future researches exploring cannabis defense pathways.

High-Fat Diet Alters the Expression of Reference Genes in Male Mice.

Quantitative PCR (qPCR), the most accurate and sensitive technique for quantifying mRNA expression, and choice of appropriate reference genes for internal error controlling in qPCR are essential to understanding the molecular mechanisms that drive the obesity epidemic and its comorbidities. In this study, using the high-fat diet (HFD)-induced obese mouse model, we assessed the expression of 10 commonly used reference genes to validate gene-expression stability in adipose tissue, liver, and muscle across different time points (4, 8, 12, and 16 weeks after HFD feeding) during the process of obesity. The data were analyzed by the GeNorm, NormFinder, BestKeeper, and Delta-Ct method, and the results showed that the most stable reference genes were different for a specific organ or tissue in a specific time point; however, PPIA, RPLP0, and YWHAZ were the top three most stable reference genes in qPCR experiments on adipose, hepatic tissues, and muscles of mice in diet-induced obesity. In addition, the mostly used genes ACTB and GAPDH were more unstable in the fat and liver, the ACTB mRNA levels were increased in four adipose tissues, and the GAPDH mRNA levels were decreased in four adipose tissues and liver after HFD feeding. These results suggest that PPIA, RPLP0, or YWHAZ may be more appropriate to be used as reference gene than ACTB and GAPDH in the adipose tissue and liver of mice during the process of high-fat diet-induced obesity.

Identification of reference genes for real-time polymerase chain reaction gene expression studies in Nile rats fed Water-Soluble Palm Fruit Extract.

The Nile rat (Arvicanthis niloticus) is a novel diurnal carbohydrate-sensitive rodent useful for studies on type 2 diabetes mellitus (T2DM) and the metabolic syndrome. Hepatic responses to T2DM and any interventions thereof can be evaluated via transcriptomic gene expression analysis. However, the study of gene expression via real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) requires identification of stably expressed reference genes for accurate normalisation. This study describes the evaluation and identification of stable reference genes in the livers from Control Nile rats as well as those supplemented with Water-Soluble Palm Fruit Extract, which has been previously shown to attenuate T2DM in this animal model. Seven genes identified as having stable expression in RNA-Sequencing transcriptome analysis were chosen for verification using real-time RT-qPCR. Six commonly used reference genes from previous literature and two genes from a previous microarray gene expression study in Nile rats were also evaluated. The expression data of these 15 candidate reference genes were analysed using the RefFinder software which incorporated analyses performed by various algorithms. The Hpd, Pnpla6 and Vpp2 genes were identified as the most stable across the 36 samples tested. Their applicability was demonstrated through the normalisation of the gene expression profiles of two target genes, Cela1 and Lepr.In conclusion, three novel reference genes which can be used for robust normalisation of real-time RT-qPCR data were identified, thereby facilitating future hepatic gene expression studies in the Nile rat.

Reference gene and tropomyosin expression in mud crab Scylla olivacea, Scylla paramamosain and Scylla tranquebarica.

Tropomyosin, a muscle tissue protein is a major allergen in most of shellfish including mud crab. Quantitative real time-PCR (qRT-PCR) using a stable reference gene is the most sensitive approach to produce accurate relative gene expression that has yet to be demonstrated for allergenic tropomyosin in mud crab species. This study was conducted to identify the suitable reference gene and tropomyosin expression in different body parts of local mud crabs, Scylla olivacea, Scylla paramamosain and Scylla tranquebarica. Myosin, 18S rRNA, GADPH and EF1α were selected as candidate reference genes and their expression was measured in the abdomen, walking leg and cheliped tissues of local Scylla spp. The expression stability was analyzed using the comparative delta-Ct method, BestKeeper, NormFinder and geNorm then comprehensively ranked by RefFinder algorithm. Findings showed that EF1α was the most suitable reference gene across three mud crab species. Meanwhile, the abdomen, walking leg and cheliped selected their own suitable reference gene either Myosin, 18S rRNA, EF1α or GADPH. Overall, tropomyosin was the highest in S. tranquebarica, whereas the least was in S. paramamosain. Interestingly, tropomyosin was the highest in the abdomen of all mud crab species. This is the first analysis on reference genes selection for qRT-PCR data normalization of tropomyosin expression in mud crab. These results will provide more accurate findings for further gene expression and allergen analysis in Scylla spp.

Selection and validation of reference genes for measuring gene expression in Toona ciliata under different experimental conditions by quantitative real-time PCR analysis

BackgroundBefore studying gene expression of different organisms, it is important to determine the best reference gene. At present, the most accurate method of detecting gene expression is quantitative real-time PCR (RT-qPCR). With this method, reference genes that are stable in different biological systems and under different conditions can be obtained. Toona ciliata Roem (T. ciliata). is a valuable and fast-growing timber specie. In this study, 20 reference genes were identified using RT-qPCR, as a primary prerequisite for future gene expression analysis. Four different methods, geNorm, NormFinder, BestKeeper, and RankAggreg were used to evaluate the expression stability of the 20 candidate reference genes in various tissues under different conditions.ResultsThe experimental results showed that TUB-α was the most stably expressed reference gene across all samples and UBC17 was the most stable in leaves and young stems under Hypsipyla robusta (H. robusta) and methyl jasmonate (MeJA) treatments. In addition, PP2C59 and UBC5B were the best-performing genes in leaves under H. robusta treatment, while HIS1 and ACT7 were the best reference genes in young stems. The two best reference genes were 60S-18 and TUB-α after treatment at 4 °C. The expression of HIS6 and MUB1 was the most stable under PEG6000 treatment. The accuracy of the selected reference genes was verified using the transcription factor MYB3 (TcMYB3) gene.ConclusionsThis is the first report to verify the best reference genes for normalizing gene expression in T. ciliata under different conditions, which will facilitate future elucidation of gene regulations in this species.

Selection and Evaluation of Optimal Reference Genes for Quantitative Reverse Transcription-Polymerase Chain Reaction Analyses of Gene Expression in Human Spermatozoa

Objective: Optimal reference genes are critical for accurate normalization and reliable interpretation of gene expression quantification data. Recently, several strategies have been utilized for validating reference genes in different human tissues. However, no universal reference genes have been described that accurately summarize transcriptional activity in human spermatozoa. Methods: Using quantitative reverse transcription-polymerase chain reaction (RT-qPCR), we evaluated ten commonly used candidate reference genes between two groups of human cryopreserved donor sperm with different pregnancy rates. We assessed the stability of reference genes using three different algorithms, namely geNorm, NormFinder, and BestKeeper. We then identified the most stable reference genes. Results: Male-enhanced antigen 1 (MEA1) was identified as the most stably expressed reference gene, followed by testis-enhanced gene transcript (TEGT). Conclusions: We comprehensively identified MEA1 and TEGT as the most stably expressed reference genes for the normalization of gene expression data in human spermatozoa.

Selecting suitable reference genes for qPCR normalization: a comprehensive analysis in MCF-7 breast cancer cell line

BackgroundMCF-7 breast cancer cell line is undoubtedly amongst the most extensively studied patient-derived research models, providing pivotal results that have over the decades translated to constantly improving patient care. Many research groups, have previously identified suitable reference genes for qPCR normalization in MCF-7 cell line. However, over the course of identification of suitable reference genes, a comparative analysis comprising these genes together in a single study has not been reported. Furthermore, the expression dynamics of these reference genes within sub-clones cultured over multiple passages (p) has attracted limited attention from research groups. Therefore, we investigated the expression dynamics of 12 previously suggested reference genes within two sub-clones (culture A1 and A2) cultured identically over multiple passages. Additionally, the effect of nutrient stress on reference gene expression was examined to postulate an evidence-based recommendation of the least variable reference genes that could be employed in future gene expression studies.ResultsThe analysis revealed the presence of differential reference gene expression within the sub-clones of MCF-7. In culture A1, GAPDH-CCSER2 were identified as the least variable reference genes while for culture A2, GAPDH-RNA28S were identified. However, upon validation using genes of interest, both these pairs were found to be unsuitable control pairs. Normalization of AURKA and KRT19 with triplet pair GAPDH-CCSER2-PCBP1 yielded successful results. The triplet also proved its capability to handle variations arising from nutrient stress.ConclusionsThe variance in expression behavior amongst sub-clones highlights the potential need for exercising caution while selecting reference genes for MCF-7. GAPDH-CCSER2-PCBP1 triplet offers a reliable alternative to otherwise traditionally used internal controls for optimizing intra- and inter-assay gene expression differences. Furthermore, we suggest avoiding the use of ACTB, GAPDH and PGK1 as single internal controls.

Validation of reference genes for expression analysis in a murine trauma model combining traumatic brain injury and femoral fracture

Systemic and local posttraumatic responses are often monitored on mRNA expression level using quantitative real-time PCR (qRT-PCR), which requires normalisation to adjust for confounding sources of variability. Normalisation requests reference (housekeeping) genes stable throughout time and divergent experimental conditions in the tissue of interest, which are crucial for a reliable and reproducible gene expression analysis. Although previous animal studies analysed reference genes following isolated trauma, this multiple-trauma gene expression analysis provides a notable study analysing reference genes in primarily affected (i.e. bone/fracture callus and hypothalamus) and secondarily affected organs (i.e. white adipose tissue, liver, muscle and spleen), following experimental long bone fracture and traumatic brain injury. We considered tissue-specific and commonly used top-ranked reference candidates from different functional groups that were evaluated applying the established expression stability analysis tools NormFinder, GeNorm, BestKeeper and RefFinder. In conclusion, reference gene expression in primary organs is highly time point as well as tissue-specific, and therefore requires careful evaluation for qRT-PCR analysis. Furthermore, the general application of Ppia, particularly in combination with a second reference gene, is strongly recommended for the analysis of systemic effects in the case of indirect trauma affecting secondary organs through local and systemic pathophysiological responses.

The Influence of Melatonin on the Daily 24-h Rhythm of Putative Reference Gene Expression in White Adipose Tissues.

In adipose tissue, the expression of hundreds of genes exhibits circadian oscillation, which may or may not be affected by circulating melatonin levels. Using control and pinealectomized rats, we investigated the daily expression profile of Actb, Hprt-1, B2m, and Rpl37a, genes that are commonly used as reference genes for reverse transcription quantitative polymerase chain reaction (RT-qPCR), in epididymal (EP), retroperitoneal (RP), and subcutaneous (SC) adipose tissues. In control rats, Actb expression presented a daily oscillation in all adipose tissues investigated, Hprt-1 showed 24-h fluctuations in only RP and SC depots, B2m was stable over 24 h for EP and RP but oscillated over 24 h in SC adipose tissue, and Rpl37a presented a daily oscillation in only RP fat. In the absence of melatonin, the rhythmicity of Actb in all adipose depots was abolished, the daily rhythmicity of Hprt-1 and B2m was disrupted in SC fat, the peak expression of Rpl37a and Hprt-1 was delayed, and the amplitude of Rpl37a was reduced in RP adipose tissue. Collectively, our results demonstrate that the expression of putative reference genes displays a daily rhythm influenced by melatonin levels in a manner specific to the adipose depot. Thus, the proper standardization and daily profile expression of reference genes should be performed carefully in temporal studies using RT-qPCR analysis.

Testing reference genes for transcript profiling in Uromyces appendiculatus during urediospore infection of common bean.

Uromyces appendiculatus is a major pathogen on common bean. Like other rust fungi, it uses effectors to influence its host plant. Effectors are assumed to possess characteristic expression profiles, reflecting their activity during the infection process. In order to determine expression profiles using RT-qPCR, stably expressed reference genes are necessary for normalization. These reference genes need to be tested. Using samples representing seven different developmental stages of the urediospore-based infection process we employed RT-qPCR to measure the expression of 14 candidate reference genes and determined the most suitable ones based on the range of Cq values and comparative calculations using the geNorm and NormFinder algorithms. Among the tested genes RPS14 had the smallest Cq range, followed by Elf1a and Elf3; geNorm rated Tub and UbcE2 best with CytB as a third and NormFinder found UbcE2, Tub and Elf3 as best reference genes. Combining these findings using equal weight for the rankings UbcE2, Elf3 and Tub can be considered the best reference genes. A combination of either two reference genes, UbcE2 and Tub or three reference genes, UbcE2, Tub, and Elf3 is recommended for normalization. However, differences between most genes were relatively small, so all tested genes can be considered suitable for normalization with the exception of RPS9, SDH, Ubc and PDK.