Potential Reference Genes Research Articles

Selection and validation of reference genes for gene expression analysis in apomictic and sexual Cenchrus ciliaris

BackgroundApomixis is a naturally occurring asexual mode of seed reproduction resulting in offspring genetically identical to the maternal plant. Identifying differential gene expression patterns between apomictic and sexual plants is valuable to help deconstruct the trait. Quantitative RT-PCR (qRT-PCR) is a popular method for analyzing gene expression. Normalizing gene expression data using proper reference genes which show stable expression under investigated conditions is critical in qRT-PCR analysis. We used qRT-PCR to validate expression and stability of six potential reference genes (EF1alpha, EIF4A, UBCE, GAPDH, ACT2 and TUBA) in vegetative and reproductive tissues of B-2S and B-12-9 accessions of C. ciliaris.FindingsAmong tissue types evaluated, EF1alpha showed the highest level of expression while TUBA showed the lowest. When all tissue types were evaluated and compared between genotypes, EIF4A was the most stable reference gene. Gene expression stability for specific ovary stages of B-2S and B-12-9 was also determined. Except for TUBA, all other tested reference genes could be used for any stage-specific ovary tissue normalization, irrespective of the mode of reproduction.ConclusionOur gene expression stability assay using six reference genes, in sexual and apomictic accessions of C. ciliaris, suggests that EIF4A is the most stable gene across all tissue types analyzed. All other tested reference genes, with the exception of TUBA, could be used for gene expression comparison studies between sexual and apomictic ovaries over multiple developmental stages. This reference gene validation data in C. ciliaris will serve as an important base for future apomixis-related transcriptome data validation.

Gene Expression Studies: How to Obtain Accurate and Reliable Data by Quantitative Real-Time RT PCR / IZUČAVANJE EKSPRESIJE GENA: KAKO DOBITI TAČNE I POUZDANE PODATKE KVANTITATIVNIM RT PCR-OM U REALNOM VREMENU

Summary Real-time RT PCR has been recognized as an accurate, reliable and sensitive method for quantifying gene transcription. However, several steps preceding PCR represent critical points and source of inaccuracies. These steps include cell processing, RNA extraction, RNA storage, assessment of RNA concentration and cDNA synthesis. To compensate for potential variability introduced by the procedure, normalization of target gene expression has been established. Accurate normalization has become an absolute prerequisite for the correct quantification of gene expression. Several strategies are in use for the normalization of data, including normalization to sample size, to total RNA or to an internal reference. Among these, the use of housekeeping genes as an internal (endogenous) control is the most common approach. Given the increased sensitivity, reproducibility and large dynamic range of this methodology, the requirements for a proper reference gene for normalization have become increasingly stringent. The aim of this paper is to discuss the concept of normalization in mRNA quantification, as well as to discuss several statistical algorithms developed to help the validation of potential reference genes. By showing that the use of inappropriate endogenous control might lead to incorrect results and misinterpretation of experimental data, we are joining the creators of Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) in an attempt to convince scientists that proper validation of potential reference genes is an absolute prerequisite for correct normalization and, therefore, for providing accurate and reliable data by quantitative real-time RT PCR gene expression analyses.

FFPE tissue as a feasible source for gene expression analysis – A comparison of three reference genes and one tumor marker

BackgroundFormalin-fixation, paraffin-embedding is the standard processing technique for tumor tissue in modern pathology. New techniques such as cryo-conservation allow rapid fixation and long-time storage but come along with increased costs and enlarged storage complexity. However, formalin-fixed, paraffin-embedded (FFPE) tissue is available in a large quantity, making it the ideal material for retrospective studies.The following study was designed to investigate the influence of formalin-fixation on the quality of mRNA and applicability of FFPE-derived mRNA for gene expression analysis. Three potential reference genes for pulmonary tumors with neuroendocrine differentiation were included and tested for their robust expression. Materials and methodsEighty specimens collected from 2005 to 2012 at the Institute of Pathology and Neuropathology at the University Hospital Essen were analyzed for their gene expression by using TaqMan® gene expression assays on demand (AoD). Three distinct potential reference genes (ACTB, GAPDH, HPRT1) were evaluated for their expression, and a proteasome subunit (PSMA1) was included in the analysis as tumor marker and functioned as an internal technical control. ConclusionFor GAPDH and ACTB, a highly significant correlation and consistent expression between the investigated entities was found, making them reliable reference genes for further research. Additionally, the feasibility for a FFPE tissue-based gene expression analysis was verified by showing that the mRNA quality is sufficient. When standardized FFPE preparation is performed carefully, sufficient mRNA can be isolated for reliable and successful gene expression analysis. That provides the basis the door for large, retrospective studies that correlate molecular and clinical follow-up data.

Evaluation of suitable reference genes for qRT-PCR gene expression normalization in reproductive, vegetative tissues and during fruit development in oil palm

Gene expression patterns of a target gene in different tissues and at different stages of development can provide clues towards the understanding of its biological function. The accuracy of gene expression via quantitative real-time reverse transcription-PCR (qRT-PCR) analysis strongly depends on transcript normalization using reference genes that demonstrate consistent expression levels in tissues and experimental conditions studied. However, suitable reference genes for qRT-PCR in oil producing fruit have not been well identified. In this study, the expression stability of fourteen potential reference genes for qRT-PCR analysis in diverse sets of biological samples including six distinct oil palm organs, two floral tissues, four vegetative tissues, five stages of fruit development and two differential phenotype fruit tissue was evaluated. The expression stabilities were assessed using three statistical algorithms including geNorm, NormFinder and qBASE plus. The stability rankings from three outputs were consolidated to obtain the consensus stability ranking of all reference genes. Our analysis showed that a Gibberellin-responsive protein (GRAS) and Cyclophilin 2 (Cyp2) were the most stable genes in oil palm tissues tested. A combination of Cyp2, GRAS and SLU7 were identified as most suitable reference genes for all oil palm tissues and vegetative tissues set while in reproductive tissues set, GRAS and Glutaredoxin was recommended. Cyp2 and GRAS were required for normalization in mesocarp tissues at various developmental stages and differential oil yielding mesocarp tissues. In addition, the relative gene expression profile of two fatty acid biosynthesis genes, EgACCase and EgSTEA was conducted to confirm the validity of the reference genes in this study. Both EgACCase and EgSTEA were analyzed in fruit tissues at various developmental stages. This study identified the most suitable reference genes for normalization of gene expression in oil palm. These results serve as a guideline for the selection of suitable reference genes under different experimental conditions and accurate normalization of gene expression studies in a wide variety of tissues in oil palm.

Validation of reference genes for quantitative gene expression studies in Volvox carteri using real-time RT-PCR

Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) is a sensitive technique for analysis of gene expression under a wide diversity of biological conditions. However, the identification of suitable reference genes is a critical factor for analysis of gene expression data. To determine potential reference genes for normalization of qRT-PCR data in the green alga Volvox carteri, the transcript levels of ten candidate reference genes were measured by qRT-PCR in three experimental sample pools containing different developmental stages, cell types and stress treatments. The expression stability of the candidate reference genes was then calculated using the algorithms geNorm, NormFinder and BestKeeper. The genes for 18S ribosomal RNA (18S) and eukaryotic translation elongation factor 1α2 (eef1) turned out to have the most stable expression levels among the samples both from different developmental stages and different stress treatments. The genes for the ribosomal protein L23 (rpl23) and the TATA-box binding protein (tbpA) showed equivalent transcript levels in the comparison of different cell types, and therefore, can be used as reference genes for cell-type specific gene expression analysis. Our results indicate that more than one reference gene is required for accurate normalization of qRT-PCRs in V. carteri. The reference genes in our study show a much better performance than the housekeeping genes used as a reference in previous studies.

Identification and Evaluation of Reference Genes for qRT-PCR Normalization in Ganoderma lucidum

Quantitative real-time reverse transcription PCR (qRT-PCR) is a rapid, sensitive, and reliable technique for gene expression studies. The accuracy and reliability of qRT-PCR results depend on the stability of the reference genes used for gene normalization. Therefore, a systematic process of reference gene evaluation is needed. Ganoderma lucidum is a famous medicinal mushroom in East Asia. In the current study, 10 potential reference genes were selected from the G. lucidum genomic data. The sequences of these genes were manually curated, and primers were designed following strict criteria. The experiment was conducted using qRT-PCR, and the stability of each candidate gene was assessed using four commonly used statistical programs-geNorm, NormFinder, BestKeeper, and RefFinder. According to our results, PP2A was expressed at the most stable levels under different fermentation conditions, and RPL4 was the most stably expressed gene in different tissues. RPL4, PP2A, and β-tubulin are the most commonly recommended reference genes for normalizing gene expression in the entire sample set. The current study provides a foundation for the further use of qRT-PCR in G. lucidum gene analysis.

Evaluation of potential reference genes for qRT-PCR studies in human hepatoma cell lines treated with TNF-α

In this study, the expression of eight candidate reference genes, B2M, ACTB, GAPDH, HMBS, HPRT1, TBP, UBC, and YWHAZ, was examined to identify optimal reference genes by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis in two human hepatoma cell lines, BEL-7402 and SMMC-7721, treated with tumor necrosis factor-α (TNF-α) for different time periods. The expression stability of these genes was analyzed by three independent algorithms: geNorm, NormFinder, and BestKeeper. Results showed that TBP was the most stably expressed gene in BEL-7402 and SMMC-7721 cell lines under current experimental conditions, and that the optimal set of reference genes required for accurate normalization was TBP and HMBS, based on the pairwise variation value determined with geNorm. UBC and ACTB were ranked as the least stable genes by same algorithms. Our findings provide evidence that using TBP alone or in combination with HMBS as endogenous controls could be a reliable method for normalizing qRT-PCR data in human hepatoma cell lines treated with TNF-α.

The choice of reference gene set for assessing gene expression in barley (Hordeum vulgare L.) under low temperature and drought stress

Drought and low temperature are the two most significant causes of abiotic stress in agricultural crops and, therefore, they pose considerable challenges in plant science. Hence, it is crucial to study response mechanisms and to select genes for identification signaling pathways that lead from stimulus to response. The assessment of gene expression is often attempted using real-time RT-PCR (qRT-PCR), a technique which requires a careful choice of reference gene(s) for normalization purpose. Here, we report a comparison of 13 potential reference genes for studying gene expression in the leaf and crown of barley seedlings subjected to low temperature or drought stress. All three currently available software packages designed to identify reference genes from qRT-PCR data (GeNorm, NormFinder and BestKeeper) were used to identify informative sets of up to three reference genes. Interestingly, the data obtained from the separate treatment of leaf and crown have led to the recommendations that HSP70 and S-AMD (and possibly HSP90) to be used as the reference genes for low-temperature stressed leaves, HSP90 and EF1α for low-temperature stressed crowns, cyclophilin and ADP-RF (and possibly ACT) for drought-stressed leaves, and EF1α and S-AMD for drought-stressed crowns. Our results have demonstrated that the gene expression can be highly tissue- or organ-specific in barley and have confirmed that reference gene choice is essential in qRT-PCR. The findings can also serve as guidelines for the selection of reference genes under different stress conditions and lay foundation for more accurate and widespread use of qRT-PCR in barley gene analysis.

Validation of Suitable Reference Genes for Expression Studies in Different Pilocarpine-Induced Models of Mesial Temporal Lobe Epilepsy

It is well recognized that the reference gene in a RT-qPCR should be properly validated to ensure that gene expression is unaffected by the experimental condition. We investigated eight potential reference genes in two different pilocarpine PILO-models of mesial temporal lobe epilepsy (MTLE) performing a stability expression analysis using geNorm, NormFinder and BestKepeer softwares. Then, as a validation strategy, we conducted a relative expression analysis of the Gfap gene. Our results indicate that in the systemic PILO-model Actb, Gapdh, Rplp1, Tubb2a and Polr1a mRNAs were highly stable in hippocampus of rats from all experimental and control groups, whereas Gusb revealed to be the most variable one. In fact, we observed that using Gusb for normalization, the relative mRNA levels of the Gfap gene differed from those obtained with stable genes. On the contrary, in the intrahippocampal PILO-model, all softwares included Gusb as a stable gene, whereas B2m was indicated as the worst candidate gene. The results obtained for the other reference genes were comparable to those observed for the systemic Pilo-model. The validation of these data by the analysis of the relative expression of Gfap showed that the upregulation of the Gfap gene in the hippocampus of rats sacrificed 24 hours after status epilepticus (SE) was undetected only when B2m was used as the normalizer. These findings emphasize that a gene that is stable in one pathology model may not be stable in a different experimental condition related to the same pathology and therefore, the choice of reference genes depends on study design.

Characterization of reference gene expression in tung tree (Vernicia fordii)

Tung oil from tung tree (Vernicia fordii) is widely used as a drying ingredient in paints, varnishes, and other coatings and finishes. Recent research has focused on the understanding of the biosynthesis of oil in tung trees. Many oil biosynthetic genes have been identified in tung tree but little is known about the expression patterns of the genes in tung seeds. Quantitative real-time-PCR (qPCR) assays are widely used for gene expression analysis. One crucial task of qPCR assay design is to select stably expressed internal reference genes for data analysis. The objective of this study was to characterize the expression of potential reference genes in the tung tree to provide a sound basis for reliable and reproducible qPCR results. The expression of tung 60s ribosome protein L19 (Rpl19b), glyceraldehyde 3 phosphate dehydrogenase (Gapdh) and ubiquitin protein ligase (Ubl) was examined by TaqMan and SYBR Green qPCR using RNA from three trees, three tissues (seeds, leaves and flowers) and 11 time events of developing seeds. The variations of the three mRNA levels were compared between two RNA extraction methods, two cDNA preparations, and the same or different PCR plates. Overall results demonstrated that Rpl19b was the most stably expressed gene, followed closely by Ubl, and Gapdh was the worst among the three genes under optimized qPCR assay conditions. These results suggest that Rpl19b and Ubl are preferable reference genes for both TaqMan and SYBR Green qPCR assays. The development of these reference genes for quantitative gene expression analyses in tung trees should facilitate identifying target genes for genetic engineering industrial oils in oilseed crops.

Selection and validation of reference genes for quantitative gene expression studies by real-time PCR in eggplant (Solanum melongena L)

BackgroundAnalysis of gene expression patterns leads to functional understanding of biological processes. Quantitative real-time PCR has become the most commonly used technique for in-depth studies of gene expression. To quantify variation in specific gene expression, accurate and reliable normalization across different samples and tissues is necessary. This can be achieved by selecting one or more suitable reference genes to compare the target mRNA transcript levels. In the present work, we illustrate the first evaluation of potential internal control or reference genes across different developmental stages of eggplant for reliable quantification of transcripts by real-time PCR.ResultsWe have evaluated the stability in expression of six candidate reference genes (18s rRNA, APRT, GAPDH, Cyclophilin, Actin, and RuBP) in a set of tissues representing six developmental stages of eggplant. The candidate genes were cloned from cDNA and analysed by real-time PCR. The expression data analyzed by three statistical methods (geNorm, NormFinder and BestKeeper) identified 18s rRNA, Cyclophilin and APRT as the most stable and suitable reference genes in eggplant. This was further confirmed in four different varieties, two representative lines of transgenic eggplant as well as in nematode infected eggplant.Conclusion18s rRNA, Cyclophilin and APRT have been found to be appropriate for the normalization of real-time PCR data for gene expression studies in eggplant.

Identification and validation of reference genes for transcript normalization in strawberry (Fragaria × ananassa) defense responses.

Strawberry (Fragaria spp) is an emerging model for the development of basic genomics and recombinant DNA studies among rosaceous crops. Functional genomic and molecular studies involve relative quantification of gene expression under experimental conditions of interest. Accuracy and reliability are dependent upon the choice of an optimal reference control transcript. There is no information available on validated endogenous reference genes for use in studies testing strawberry-pathogen interactions. Thirteen potential pre-selected strawberry reference genes were tested against different tissues, strawberry cultivars, biotic stresses, ripening and senescent conditions, and SA/JA treatments. Evaluation of reference candidate’s suitability was analyzed by five different methodologies, and information was merged to identify best reference transcripts. A combination of all five methods was used for selective classification of reference genes. The resulting superior reference genes, FaRIB413, FaACTIN, FaEF1α and FaGAPDH2 are strongly recommended as control genes for relative quantification of gene expression in strawberry. This report constitutes the first systematic study to identify and validate optimal reference genes for accurate normalization of gene expression in strawberry plant defense response studies.

Reverse transcription quantitative real-time polymerase chain reaction reference genes in the spared nerve injury model of neuropathic pain: validation and literature search

BackgroundThe reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a widely used, highly sensitive laboratory technique to rapidly and easily detect, identify and quantify gene expression. Reliable RT-qPCR data necessitates accurate normalization with validated control genes (reference genes) whose expression is constant in all studied conditions. This stability has to be demonstrated.We performed a literature search for studies using quantitative or semi-quantitative PCR in the rat spared nerve injury (SNI) model of neuropathic pain to verify whether any reference genes had previously been validated. We then analyzed the stability over time of 7 commonly used reference genes in the nervous system – specifically in the spinal cord dorsal horn and the dorsal root ganglion (DRG). These were: Actin beta (Actb), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal proteins 18S (18S), L13a (RPL13a) and L29 (RPL29), hypoxanthine phosphoribosyltransferase 1 (HPRT1) and hydroxymethylbilane synthase (HMBS). We compared the candidate genes and established a stability ranking using the geNorm algorithm. Finally, we assessed the number of reference genes necessary for accurate normalization in this neuropathic pain model.ResultsWe found GAPDH, HMBS, Actb, HPRT1 and 18S cited as reference genes in literature on studies using the SNI model. Only HPRT1 and 18S had been once previously demonstrated as stable in RT-qPCR arrays. All the genes tested in this study, using the geNorm algorithm, presented gene stability values (M-value) acceptable enough for them to qualify as potential reference genes in both DRG and spinal cord. Using the coefficient of variation, 18S failed the 50% cut-off with a value of 61% in the DRG. The two most stable genes in the dorsal horn were RPL29 and RPL13a; in the DRG they were HPRT1 and Actb. Using a 0.15 cut-off for pairwise variations we found that any pair of stable reference gene was sufficient for the normalization process.ConclusionsIn the rat SNI model, we validated and ranked Actb, RPL29, RPL13a, HMBS, GAPDH, HPRT1 and 18S as good reference genes in the spinal cord. In the DRG, 18S did not fulfill stability criteria. The combination of any two stable reference genes was sufficient to provide an accurate normalization.

Validation of Reliable Reference Genes for Real-Time PCR in Human Umbilical Vein Endothelial Cells on Substrates with Different Stiffness

BackgroundThe mechanical properties of cellular microenvironments play important roles in regulating cellular functions. Studies of the molecular response of endothelial cells to alterations in substrate stiffness could shed new light on the development of cardiovascular disease. Quantitative real-time PCR is a current technique that is widely used in gene expression assessment, and its accuracy is highly dependent upon the selection of appropriate reference genes for gene expression normalization. This study aimed to evaluate and identify optimal reference genes for use in studies of the response of endothelial cells to alterations in substrate stiffness.Methodology/Principal FindingsFour algorithms, GeNormPLUS, NormFinder, BestKeeper, and the Comparative ΔCt method, were employed to evaluate the expression of nine candidate genes. We observed that the stability of potential reference genes varied significantly in human umbilical vein endothelial cells on substrates with different stiffness. B2M, HPRT-1, and YWHAZ are suitable for normalization in this experimental setting. Meanwhile, we normalized the expression of YAP and CTGF using various reference genes and demonstrated that the relative quantification varied according to the reference genes.Conclusion/Significance:Consequently, our data show for the first time that B2M, HPRT-1, and YWHAZ are a set of stably expressed reference genes for accurate gene expression normalization in studies exploring the effect of subendothelial matrix stiffening on endothelial cell function. We furthermore caution against the use of GAPDH and ACTB for gene expression normalization in this experimental setting because of the low expression stability in this study.

Selection of Internal Control Genes for Real-Time Quantitative PCR in Ovary and Uterus of Sows across Pregnancy

BackgroundReproductive traits play a key role in pig production in order to reduce costs and increase economic returns. Among others, gene expression analyses represent a useful approach to study genetic mechanisms underlying reproductive traits in pigs. The application of reverse-transcription quantitative PCR requires the selection of appropriate reference genes, whose expression levels should not be affected by the experimental conditions, especially when comparing gene expression across different physiological stages.ResultsThe gene expression stability of ten potential reference genes was studied by three different methods (geNorm, NormFinder and BestKeeper) in ovary and uterus collected at five different physiological time points (heat, and 15, 30, 45 and 60 days of pregnancy). Although final ranking differed, the three algorithms gave very similar results. Thus, the most stable genes across time were TBP and UBC in uterus and TBP and HPRT1 in ovary, while HMBS and ACTB showed the less stable expression in uterus and ovary, respectively. When studied as a systematic effect, the reproductive stage did not significantly affect the expression of the candidate reference genes except at 30d and 60d of pregnancy, when a general drop in expression was observed in ovary.ConclusionsBased in our results, we propose the use of TBP, UBC and SDHA in uterus and TBP, GNB2L1 and HPRT1 in ovary for normalization of longitudinal expression studies using quantitative PCR in sows.

Reference Gene Selection for Quantitative PCR Studies in Sheep Neutrophils

Reference genes are essential for studying mRNA expression with quantitative PCR (qPCR). We investigated 11 potential neutrophil reference genes (RPL19, GAPDH, ACTB, B2M, HPRT, G6PD, TFRC, PGK1, YWHAZ, SDHA and GYPC) for sheep under disease conditions of foot rot (FR) and with or without Se supplementation. Initial screening was based on gene expression level (<28 Cq cycles) and variability (SD < 1.5 Cq cycles) and excluded TFRC, GYPC and HPRT from further analysis. Expression stability of the remaining genes was evaluated using four software programs: geNorm, NormFinder, BestKeeper and the comparative delta Cq method. The neutrophil reference genes, G6PD, YWHAZ, GAPDH, RPL19 and SDHA, consistently ranked among the top five most stable genes under these experimental conditions. The SDHA gene expression was not stable in FR-diseased sheep receiving Se treatment and, thus, cannot be recommended as a reference gene. The commonly used genes, PGK1, ACTB and B2M, were not reliable reference genes, underscoring the need to validate neutrophil reference genes under different experimental conditions. Multiple references genes rather than a single gene may provide more robust and reliable results. The best pair of reference genes was SDHA/G6PD in healthy sheep and GADPH/YWHAZ in FR-diseased sheep.

Identification and validation of reference genes for gene expression studies in postharvest rose flower (Rosa hybrida)

Optimal reference genes are important for data normalization so that accurate and reliable gene expression measurements may be obtained in both semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR (qRT-PCR) methods. This study firstly evaluated potential reference genes in petals of rose flower (Rosa hybrida) under postharvest stress conditions and in various floral organs during flower opening and senescence, combining both RT-PCR and qRT-PCR analysis. The expression stabilities of gene members from three traditional housekeeping gene families – actin (RhACT), tubulin (RhTUB) and ubiquitin (RhUBI) – were assessed using two analysis software packages, geNorm and NormFinder. The results showed that, for cut rose flower, the optimal reference genes were RhUBI1 for dehydration treatment and receptacles; RhTUB2 for exogenous ethylene; RhACT4 for gibberellic and abscisic acid treatments, wounding and stamens; RhUBI6 for petals; RhUBI2 for sepals; and RhACT1 for gynoecia, respectively. Our results provide guidelines for reference gene(s) selection under different postharvest conditions and point the way towards more accurate and widespread use of qRT-PCR in rose flower.

Comparative analysis of reference gene stability in human mesenchymal stromal cells during osteogenic differentiation

Mesenchymal stromal cells (MSCs) are one of the most frequently used cell sources for tissue engineering strategies. Cultivation of osteogenic MSCs is a prerequisite for cell-based concepts that aim at bone regeneration. Quantitative real time reverse transcription polymerase chain reaction (qRT-PCR) analysis is a commonly used method for the examination of mRNA expression levels. However, data on suitable reference genes for osteogenically cultivated MSCs is scarce. Hence, the aim of the study was to compare the regulation of different potential reference genes in osteogenically stimulated MSCs. Human MSCs were isolated from bone marrow aspirates of N = 6 hematologically healthy individuals, expanded by polystyrene-adherence, and maintained with and without osteogenic supplements for 14 days. Cellular proliferation and osteogenic differentiation were assessed by total DNA quantification, cell-specific alkaline phosphatase (ALP) activity and by qualitative staining for ALP and alizarin red, respectively. mRNA expression levels of N = 32 potential reference genes were quantified using the human Endogenous Control TaqMan® assays. mRNA expression stability was calculated using geNorm. The combined use of the most stable reference genes and DNA-damage-inducible alpha, Pumilio homolog 1, and large ribosomal protein P0 significantly improved gene expression accuracy as compared to the use of the commonly used reference genes beta actin and glyceraldehyde-3-phosphate dehydrogenase during qRT-PCR-based target gene expression analysis of osteogenically stimulated MSCs.

Evaluation of potential reference genes for use in gene expression studies in the conifer pathogen (Heterobasidion annosum)

The basidiomycete Heterobasidion annosum is the causative agent of butt and root rot disease of conifer trees and it's one of the most destructive conifer pathogen in the northern hemisphere. Because of the intrinsic difficulties in genome manipulation in this fungus, most studies have been focused on gene expression analysis using quantitative real time polymerase chain reaction (qPCR). qPCR is a powerful technique but its reliability resides in the correct selection of a set of reference genes used in the data normalization. In this study, we determined the expression stability of 11 selected reference genes in H. annosum. Almost nothing has so far been published about validation of a set of reference genes to be used in gene expression experiments in this fungus. Eleven reference genes were validated in H. annosum which was grown on three different substrates: pine bark, pine heartwood, and pine sapwood. Bestkeeper and NormFinder Excel-based software were used to evaluate the reference gene transcripts' stability. The results from these two programs indicated that three reference genes namely Tryp metab, RNA Pol3 TF, and Actin were stable in H. annosum in the conditions studied. Interestingly, the GAPDH transcript which has been extensively used in qPCR data normalization is not the best choice when a wide reference gene selection is available. This work represents the first extensive validation of reference genes in H. annosum providing support for gene expression studies and benefits for the wider forest pathology community.

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

Rice (Oryza sativa L.) is important to food security and is also an excellent model plant for numerous cereal crops. A functional genomics study in rice includes characterization of the expression dynamics of genes by quantitative real-time PCR (qPCR) analysis; this is a significant key for developing rice varieties that perform well in the face of adverse climate change. The qPCR analysis requires the use of appropriate reference genes in order to make any quantitative interpretations meaningful. Here, the new potential reference genes were selected from a huge public database of rice microarray experiments. The expression stability of 14 candidates and 4 conventional reference genes was validated by geNormPLUS and NormFinder software. Seven candidates are superior to the conventionally used reference genes in qPCR and three genes can be used reliably for quantitating the expression of genes involved in abiotic stress responses. These high-quality references EP (LOC_Os05g08980), HNR (LOC_Os01g71770), and TBC (LOC_Os09g34040) worked very well in three indica genotypes and one japonica genotype. One of indica genotypes including the Jasmine rice, KDML105 developed in Thailand for which no reference genes have been reported until now.