Optimal Reference Genes for Gene Expression Normalization in Trichomonas vaginalis.

Trichomonas vaginalis is the etiologic agent of trichomonosis, the most common non-viral sexually transmitted disease worldwide. This infection is associated with several health consequences, including cervical and prostate cancers and HIV acquisition. Gene expression analysis has been facilitated because of available genome sequences and large-scale transcriptomes in T. vaginalis, particularly using quantitative real-time polymerase chain reaction (qRT-PCR), one of the most used methods for molecular studies. Reference genes for normalization are crucial to ensure the accuracy of this method. However, to the best of our knowledge, a systematic validation of reference genes has not been performed for T. vaginalis. In this study, the transcripts of nine candidate reference genes were quantified using qRT-PCR under different cultivation conditions, and the stability of these genes was compared using the geNorm and NormFinder algorithms. The most stable reference genes were α-tubulin, actin and DNATopII, and, conversely, the widely used T. vaginalis reference genes GAPDH and ß-tubulin were less stable. The PFOR gene was used to validate the reliability of the use of these candidate reference genes. As expected, the PFOR gene was upregulated when the trophozoites were cultivated with ferrous ammonium sulfate when the DNATopII, α-tubulin and actin genes were used as normalizing gene. By contrast, the PFOR gene was downregulated when the GAPDH gene was used as an internal control, leading to misinterpretation of the data. These results provide an important starting point for reference gene selection and gene expression analysis with qRT-PCR studies of T. vaginalis.

Identification of optimal reference genes for gene expression normalization in a wide cohort of endometrioid endometrial carcinoma tissues.

Accurate normalization is a primary component of a reliable gene expression analysis based on qRT-PCR technique. While the use of one or more reference genes as internal controls is commonly accepted as the most appropriate normalization strategy, many qPCR-based published studies still contain data poorly normalized and reference genes arbitrarily chosen irrespective of the particular tissue and the specific experimental design. To date, no validated reference genes have been identified for endometrial cancer tissues. In this study, 10 normalization genes (GAPDH, B2M, ACTB, POLR2A, UBC, PPIA, HPRT1, GUSB, TBP, H3F3A) belonging to different functional and abundance classes in various tissues and used in different studies, were analyzed to determine their applicability. In total, 100 endometrioid endometrial cancer samples, which were carefully balanced according to their tumor grade, and 29 normal endometrial tissues were examined using SYBR Green Real-Time RT-PCR. The expression stability of candidate reference genes was determined and compared by means of geNorm and NormFinder softwares. Both algorithms were in agreement in identifying GAPDH, H3F3A, PPIA, and HPRT1 as the most stably expressed genes, only differing in their ranking order. Analysis performed on the expression levels of all candidate genes confirm HPRT1 and PPIA as the most stably expressed in the study groups regardless of sample type, to be used alone or better in combination. As the stable expression of HPRT1 and PPIA between normal and tumor endometrial samples fulfill the basic requirement of a reference gene to be used for normalization purposes, HPRT1 expression showed significant differences between samples from low-grade and high-grade tumors. In conclusion, our results recommend the use of PPIA as a single reference gene to be considered for improved reliability of normalization in gene expression studies involving endometrial tumor samples at different tumor degrees.

Reference gene selection for quantitative real-time polymerase chain reaction in Populus.

Accurate quantification of gene expression with quantitative real-time polymerase chain reaction (qRT-PCR) relies on the choice of an appropriate reference gene. In this study, nine candidate reference genes were selected to study the expression stability for qRT-PCR normalization in adventitious rooting of Populus hardwood cuttings. geNorm, NormFinder, and BestKeeper analysis revealed that actin isoform B (ACT) was the most unstable gene across developmental stages, whereas elongation factor 1 alpha (EF1a) and 18S recombinant RNA (18S) emerged as the most appropriate reference genes for qRT-PCR analysis in this complex developmental process.

Selection of reference genes for gene expression studies in astrocytomas.

This study was aimed to test a panel of six housekeeping genes (GAPDH, HPRT1, POLR2A, RPLP0, ACTB, and H3F) so as to identify and validate the most suitable reference genes for expression studies in astrocytomas. GAPDH was the most stable and HPRT1 was the least stable reference gene. The effect of reference gene selection on quantitative real-time polymerase chain reaction data interpretation was demonstrated, normalizing the expression data of a selected gene of interest. Thus, GAPDH may be recommended for data normalization in gene expression studies in astrocytomas. Nevertheless, a preliminary validation of reference gene stability is required prior to every study.

Evaluation of potential reference genes for real time RT-PCR studies in Atlantic halibut (Hippoglossus Hippoglossus L.); during development, in tissues of healthy and NNV-injected fish, and in anterior kidney leucocytes.

BACKGROUND: Real time RT-PCR has become an important tool for analyzing gene expression in fish. Although several housekeeping genes have been evaluated in Atlantic halibut (Hippoglossus Hippoglossus L.), appropriate reference genes for low copy mRNA transcripts at the earliest developmental stages have not been identified. No attempts have been reported to identify suitable reference genes in halibut infected with NNV or in stimulated halibut leucocytes. In this study, beta-actin1 (ACTB1), elongation factor 1 alpha (EF1A1), hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1), ribosomal protein L7 (RPL7), tubulin beta 2C (Tubb2C), and ubiquitin-conjugating enzyme (UbcE) were evaluated as reference genes for normalization of real time RT-PCR data during Atlantic halibut development, in tissue of healthy and NNV-infected fish, and in in vivo and in vitro stimulated anterior kidney leucocytes. RESULTS: The expression of all six genes was relatively stable from the unfertilized egg until 12 day degrees post fertilization (ddpf). However, none of the selected genes were found to be stably expressed throughout halibut development. The mRNA levels of the six genes increased from 18 ddpf, when zygotic transcription is likely to be activated, and stabilized at different time points. The Excel-based software programs BestKeeper, geNorm, and NormFinder ranked EF1A1 and UbcE as the best candidate reference genes before activation of zygotic transcription, and RPL7 and EF1A1 as the best candidates after hatching. EF1A1 and RPL7 were also listed as the best reference genes when exploring the expression levels of the six genes in various halibut organs, both in non-injected fish and in mock- and NNV-injected fish. None of the reference genes were found optimal for normalization of real time RT-PCR data from in vitro stimulated anterior kidney leucocytes. CONCLUSION: Generally, it was found that EF1A1 and RPL7 were the genes that showed least variation, with HPRT1 and UbcE as intermediate genes, and ACTB1 and Tubb2C as the least stable ones. None of the six reference genes can be recommended as reference gene candidates in ConA-PMA stimulated leucocytes. However, UbcE can be a good candidate in other experimental setups. This study emphasizes the need for reference gene evaluation, as universal reference genes have not been identified.

Candidate reference genes for gene expression studies in water lily.

The selection of an appropriate reference gene(s) is a prerequisite for the proper interpretation of quantitative Real-Time polymerase chain reaction data. We report the evaluation of eight candidate reference genes across various tissues and treatments in the water lily by the two software packages geNorm and NormFinder. Across all samples, clathrin adaptor complexes medium subunit (AP47) and actin 11 (ACT11) emerged as the most suitable reference genes. Across different tissues, ACT11 and elongation factor 1-alpha (EF1alpha) exhibited a stable expression pattern. ACT11 and AP47 also stably expressed in roots subjected to various treatments, but in the leaves of the same plants the most stably expressed genes were ubiquitin-conjugating enzyme 16 (UBC16) and ACT11.

Validation of endogenous reference genes for qRT-PCR analysis of human visceral adipose samples.

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.

Autoinduction of androgen receptor mRNA in primary cultures of hamster (Mesocricetus auratus) harderian gland cells.

The Harderian gland (hg) is a gland which occupies a large portion of the orbital cavity. In many species, a sexual dimorphism occurs, which suggests a gonadal steroid control of the hg. The present study examines, in primary cultures of hamster hg cells, the regulation of the androgen receptor mRNA (AR mRNA) expression. In dose-response experiments measuring the expression of AR mRNA, testosterone (T) (10(-12) M) induced a 1-fold increase of AR mRNA compared with unexposed cells, and this effect reached its zenith (6.2-fold) when cells were exposed to 10(-8) M T. In other experiments, cells were exposed or not to different drugs [T, T + flutamide (F), F, T + cycloheximide (Cy), Cy] for different times (up to 96 hr). These experiments showed a time-dependent increase of AR mRNA in the cells exposed to T, while in the cells exposed to F, T + F, T + Cy, Cy, and control (unexposed), a time-dependent decrease of AR mRNA was observed. The size of the hamster AR mRNA in these in vitro experiments was similar to that observed in other mammals (9.5 kb). It is concluded that primary cultures of hamster hg cells are a valuable model for studying hg cell activity and that in this system T autoinduces its own receptor.

Evaluation of an enzyme-linked immunosorbent assay to actin in cultured fibroblasts.

The amount of actin in cultured human skin fibroblasts was determined by an enzyme-linked immunosorbent assay. Absorbance values (OD), resulting from specific binding of an antiactin monoclonal antibody to intracellular actin, were converted to cell concentrations (pg/cell) from an actin standard curve. Actin concentration for cultured human skin fibroblasts by this technique was estimated to be 55.5 picograms/cell and constituted approximately 3.9% of the total cellular protein. This assay procedure offers the following advantages: (1) It is time efficient, can be completed in 2-3 hours; (2) cells are unaltered except for membrane permeabilization; (3) the sensitivity is equal to or greater than previous procedures involving gel electrophoresis; and (4) the assay is easy and inexpensive to perform.