Potential Reference Genes Research Articles

Reference gene selection for quantitative real-time polymerase chain reaction analysis in Bombyx mori nucleopolyhedrovirus-infected silkworms

Bombyx mori nucleopolyhedrovirus (BmNPV) is the most serious viral disease in silkworms. To investigate the mechanisms of the immune responses of B. mori to a BmNPV infection, a suitable reference gene (RG) is necessary for normalizing data when studying the expression of genes in BmNPV-infected silkworms or cells. Thus, quantitative real-time PCR polymerase chain reaction was used to compare the stability of expression of nine potential RGs, including the actin A3, translation initiation factor 3 (TIF-3), TIF-A4, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18S RNA, 28S RNA, TATA-binding protein (TBP), ribosomal protein L3 (Rpl3), and α-tubulin genes, in silkworms infected with BmNPV. The results were analyzed by BestKeeper, geNorm, and NormFinder software. Overall, α-tubulin exhibited the most stable gene expression in BmNPV-infected silkworms, and this was verified by western blotting of the α-tubulin protein. Moreover, we detected the expression of some genes involved in the immune signaling pathways of silkworms after BmNPV infection using α-tubulin as an internal RG.

Reference genes for gene expression studies in the mouse heart

To be accurate, quantitative Polymerase Chain Reaction (qPCR) studies require a set of stable reference genes for normalization. This is especially critical in cardiac research because of the diversity of the clinical and experimental conditions in the field. We analyzed the stability of previously described as potential reference genes in different subsets of cardiac tissues, each representing a different field in cardiac research. The qPCR dataset was based on 119 different tissue samples derived from cardiac development to pathology in mouse adult hearts. These samples were grouped into 47 tissue types. The stability of 9 candidate genes was analyzed in each of 12 experimental conditions comprising different groupings of these tissue types. Expression stability was determined with the geNorm module of qbase+. This analysis showed that different sets of two or three reference genes are required for analysis of qPCR data in different experimental conditions in murine cardiac research.

Selection of Reference Genes for qRT-PCR Analysis of Gene Expression in Stipa grandis during Environmental Stresses.

Stipa grandis P. Smirn. is a dominant plant species in the typical steppe of the Xilingole Plateau of Inner Mongolia. Selection of suitable reference genes for the quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) is important for gene expression analysis and research into the molecular mechanisms underlying the stress responses of S. grandis. In the present study, 15 candidate reference genes (EF1 beta, ACT, GAPDH, SamDC, CUL4, CAP, SNF2, SKIP1, SKIP5, SKIP11, UBC2, UBC15, UBC17, UCH, and HERC2) were evaluated for their stability as potential reference genes for qRT-PCR under different stresses. Four algorithms were used: GeNorm, NormFinder, BestKeeper, and RefFinder. The results showed that the most stable reference genes were different under different stress conditions: EF1beta and UBC15 during drought and salt stresses; ACT and GAPDH under heat stress; SKIP5 and UBC17 under cold stress; UBC15 and HERC2 under high pH stress; UBC2 and UBC15 under wounding stress; EF1beta and UBC17 under jasmonic acid treatment; UBC15 and CUL4 under abscisic acid treatment; and HERC2 and UBC17 under salicylic acid treatment. EF1beta and HERC2 were the most suitable genes for the global analysis of all samples. Furthermore, six target genes, SgPOD, SgPAL, SgLEA, SgLOX, SgHSP90 and SgPR1, were selected to validate the most and least stable reference genes under different treatments. Our results provide guidelines for reference gene selection for more accurate qRT-PCR quantification and will promote studies of gene expression in S. grandis subjected to environmental stress.

Selection of Reliable Reference Genes for Quantitative Real-Time PCR in Golden-Line Barbell (Sinocyclocheilus grahami) During Juvenile and Adult Stages

In order to obtain reliable results of gene expression, appropriate internal control gene(s) were required for normalization before real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Here, we selected eight candidate housekeeping genes as the potential reference genes in normalizing qRT-PCR data in ten tissues of the juvenile and adult stages of a naturally tetraploid cyprinid fish Sinocyclocheilus grahami. Before qRT-PCR, appropriate primers were designed and compared for distinguishing different copies in some duplicate genes. Candidate reference genes were evaluated for their stability using online software RefFinder, which is integrated by four normal software of the comparative delta-CT, BestKeeper, NormFinder and GeNorm. According to the rankings produced by these analyses, Eef2, ACTB and G6PD were the most stable reference genes as internal controls for qRT-PCR in juvenile, while B2M, Eef2 and RPL17 were shown to be most appropriate in adult fish. All the analyses show that GAPDH was the least stable expression gene which would be unsuitable as reference gene at both stages. This is the first report about reference gene selection in Sinocyclocheilus and we think that is beneficial for the future gene expression studies in golden-line barbel.

130 A CATALOG OF REFERENCE GENES WITH HIGH, MEDIUM, AND LOW LEVELS OF EXPRESSION DURING BOVINE IN VIVO PRE-IMPLANTATION DEVELOPMENT

Appropriate reference genes for accurate normalization in RT-PCR are essential for the study of gene expression. Ideal reference genes should not only have stable expression across stages of embryo development, but also be expressed at comparable levels to the target genes. Using RNA-seq data from in vivo-produced bovine oocytes and embryos from the 2-cell to blastocyst stage (Jiang et al., 2014 BMC Genomics 15, 756), we tried to establish a catalogue of all reference genes for RT-PCR analysis. One-way ANOVA generated 4055 genes that did not differ across stages. To reduce this list, we used the entire RNA-seq data set and first removed genes with a FPKM (fragments per kilobase of transcript per million mapped reads) of <1, and then rescaled each gene’s expression values within a range of 0 to 1. We subsequently calculated the expression variance for each gene across all stages. By assuming that the calculated variances follow a Gaussian distribution and that the majority of the genes do not have a stable expression level, a gene was classified as a reference if its variance significantly deviated (P < 0.05) from these assumptions. We identified 346 potential reference genes, all of which were among the candidates from the ANOVA analysis. We arbitrarily assigned genes in this list to high (FPKM ≥ 100), medium (10 < FPKM < 100), and low expression levels (FPKM ≤ 10), and 37, 154, and 155 genes, respectively, fell into these groups. Surprisingly, none of the commonly used reference genes, such as GAPDH, PPIA, ACTB, PRL15, GUSB, and H3F2A, were identified as being stably expressed across in vivo development. This is consistent with findings of prior RT-PCR studies (Robert et al. 2002 Biol. Reprod. 67, 1465–1472; Ross et al. 2010 Cell Reprogram. 12, 709–717). The following gene ontology terms were significantly enriched for the 346 genes: cell cycle, translation, transport, chromatin, cell division, and metabolic process, indicating that the early embryos maintained constant levels of genes involved in fundamental biological functions. Finally, we performed RT-PCR to validate the RNA-seq results using different bovine in vivo-derived oocytes and embryos (n = 3/stage). We successfully validated 10 selected genes, including those in the high (CS, PGD, and ACTR3), medium (CCT5, MRPL47, COG2, CRT9, and HELLS), and low expression groups (CDC23 and TTF1). In conclusion, we recommend the use of reference genes that are expressed at comparable levels to target genes. This study offers a useful resource to aid in the appropriate selection of reference genes, which will improve the accuracy of quantitative gene expression analyses across bovine embryo pre-implantation development.

Selection of reference genes for quantitative real-time PCR in Casuarina equisetifolia under salt stress

Real time quantitative PCR (qPCR) is widely used in gene expression analysis for its accuracy and sensitivity. Reference genes serving as endogenous controls are necessary for gene normalization. In order to select an appropriate reference gene to normalize gene expression in Casuarina equisetifolia under salt stress, 10 potential reference genes were evaluated using real time qPCR in the leaves and roots of plants grown under different NaCl concentrations and treatment durations. GeNorm, NormFinder, and BestKeeper analyses reveal that elongation factor 1-alpha (EF1α) and ubiquitin-conjugating enzyme E2 (UBC) were the most appropriate reference genes for real time qPCR under salt stress. However, β-tubulin (βTUB) and actin 7, which were widely used as reference genes in other plant species, were not always stably expressed. The combination of EF1α, UBC, uncharacterized protein 2, DNAJ homolog subfamily A member 2, and glyceraldehyde-3-phosphate dehydrogenase should be ideal reference genes for normalizing gene expression data in all samples under salt stress. It indicates the need for reference gene selection for normalizing gene expression in C. equisetifolia. In addition, the suitability of reference genes selected was confirmed by validating the expression of WRKY29-like and expansin-like B1. The results enable analysis of salt response mechanism and gene expression in C. equisetifolia.

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.Material and methodsForty-three representative FFPE-specimens were used for mRNA expression analysis using the digital nCounter technology (NanoString). Based on recent literature, a total of 91 mRNA targets were investigated as potential tumor markers or reference genes. The geNorm, NormFinder algorithms and coefficient of correlation were used to identify the most stable reference genes. Statistical analysis was performed by using the R programming environment (version 3.1.1)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.

Selection of Reliable Reference Genes for Gene Expression Studies on Rhododendron molle G. Don.

The quantitative real-time polymerase chain reaction (qRT-PCR) approach has become a widely used method to analyze expression patterns of target genes. The selection of an optimal reference gene is a prerequisite for the accurate normalization of gene expression in qRT-PCR. The present study constitutes the first systematic evaluation of potential reference genes in Rhododendron molle G. Don. Eleven candidate reference genes in different tissues and flowers at different developmental stages of R. molle were assessed using the following three software packages: GeNorm, NormFinder, and BestKeeper. The results showed that EF1-α (elongation factor 1-alpha), 18S (18s ribosomal RNA), and RPL3 (ribosomal protein L3) were the most stable reference genes in developing rhododendron flowers and, thus, in all of the tested samples, while tublin (TUB) was the least stable. ACT5 (actin), RPL3, 18S, and EF1-α were found to be the top four choices for different tissues, whereas TUB was not found to favor qRT-PCR normalization in these tissues. Three stable reference genes are recommended for the normalization of qRT-PCR data in R. molle. Furthermore, the expression profiles of RmPSY (phytoene synthase) and RmPDS (phytoene dehydrogenase) were assessed using EF1-α, 18S, ACT5, RPL3, and their combination as internals. Similar trends were found, but these trends varied when the least stable reference gene TUB was used. The results further prove that it is necessary to validate the stability of reference genes prior to their use for normalization under different experimental conditions. This study provides useful information for reliable qRT-PCR data normalization in gene studies of R. molle.

Identification of reference genes for quantitative real-time PCR studies in human cell lines under copper and zinc exposure.

Accurate quantification depends on normalization of the measured gene expression data. In particular, gene expression studies with exposure to metals are challenging due their toxicity and redox-active properties. Here, we assessed the stability of potential reference genes in three cell lines commonly used to study metal cell metabolism: Caco-2 (colon), HepG2 (liver) and THP-1 (peripheral blood) under copper (Cu) or zinc (Zn) exposure. We used combined statistical tools to identify the best reference genes from a set of eleven candidates, which included traditional "housekeeping" genes such as GAPDH and B-ACTIN, in cell lines exposed to high and low, Zn and Cu concentrations. The expression stabilities of ATP5B (ATP synthase) and CYC1 (subunits of the cytochrome) were the highest considering the effect of Zn and Cu treatments whereas SDHA (succinate dehydrogenase) was found to be the most unstable gene. Even though the transcriptional effect of Zn and Cu is very different in term of redox properties, the same best reference genes were identified when Zn or Cu treatments were analyzed together. Our results indicate that ATP5B/CYC1 are the best candidates for reference genes after metal exposure, which can be used as a suitable starting point to evaluate gene expression with other metals or in different cell types in human models.

Development of a multitissue microfluidic array for assessing changes in gene expression associated with channel catfish appetite, growth, metabolism, and intestinal health

Large-scale, gene expression profiling methods allow for high throughput analysis of physiological pathways at a fraction of the cost of individual gene expression analysis. Systems, such as the Fluidigm quantitative PCR array described here, can provide powerful assessments of the effects of diet, environment, and management on physiological pathways affecting production parameters. A targeted microfluidic PCR array was designed and validated, for channel catfish (Ictalurus punctatus) representing key pathways involved in appetite, growth, metabolism, and intestinal inflammation for their potential to provide insight into the effects of diet and dietary supplements on these important physiological processes regulating feed efficiency and growth. With few exceptions, PCR primers were designed from Ictaluridae gene sequences published in GenBank. PCR amplicons from primers designed outside of Ictaluridae were sequenced to verify gene identity. All target gene primers were initially validated via conventional real-time qPCR (RT-qPCR). Combined hypothalamus/pituitary, hepatic, stomach, and intestinal tissue were used validate a 48.48 microfluidic PCR array to analyze multitissue gene expression. Use of the Fluidigm array resulted in reliable cycle threshold levels (Ct), efficiencies (E), and quality threshold scores (QS) for all but eight genes examined. Of the potential reference genes included in the panel, alpha-tubulin (TUBA) had a high QS, E, and acceptable Ct. The high throughput application of this technology, relative to conventional RT-qPCR, for assessing dietary effects on these pathways is demonstrated. Development of this targeted multi-tissue microfluidic array paves the way for the rapid evaluation of regulatory pathways in response to alternative feeding strategies, dietary formulations, and supplementation, as well as environmental and management effects for improving channel catfish culture and validates a cost-effective, dynamic, gene expression platform for use with other cultured fishes.

RPL13A and EEF1A1 Are Suitable Reference Genes for qPCR during Adipocyte Differentiation of Vascular Stromal Cells from Patients with Different BMI and HOMA-IR.

Real-time or quantitative PCR (qPCR) is a useful technique that requires reliable reference genes for data normalization in gene expression analysis. Adipogenesis is among the biological processes suitable for this technique. The selection of adequate reference genes is essential for qPCR gene expression analysis of human Vascular Stromal Cells (hVSCs) during their differentiation into adipocytes. To the best of our knowledge, there are no studies validating reference genes for the analyses of visceral and subcutaneous adipose tissue hVSCs from subjects with different Body Mass Index (BMI) and Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) index. The present study was undertaken to analyze this question. We first analyzed the stability of expression of five potential reference genes: CYC, GAPDH, RPL13A, EEF1A1, and 18S ribosomal RNA, during in vitro adipogenic differentiation, in samples from these types of patients. The expression of RPL13A and EEF1A1 was not affected by differentiation, thus being these genes the most stable candidates, while CYC, GAPDH, and 18S were not suitable for this sort of analysis. This work highlights that RPL13A and EEF1A1 are good candidates as reference genes for qPCR analysis of hVSCs differentiation into adipocytes from subjects with different BMI and HOMA-IR.

Normalization of qPCR in platelets – YWHAE a potential genericreference gene

The mRNA of human platelets has been extensively studied and it is generally appreciated that platelets contain mRNA transcripts derived from the megakaryocytes, and they have the ability to translate it into proteins. Additionally, platelets contain microRNA (miRNA) that has been shown to potentially regulate the translation of certain proteins. When quantifying gene expression by quantitative real-time polymerase chain reaction (qPCR), a valid normalization method is required and the use of reference genes is a common and robust approach. It is recommended to perform a proper validation of potential reference genes for each individual experimental setup. Previous studies have mainly been performed using commonly used reference genes for nucleated cells, and to our knowledge there are no global evaluations of the stability of transcripts in platelets. Finding a stable transcript would be valuable for inter-study comparisons, and the aim of this study was to identify one or more stable mRNA transcripts suitable as generic reference genes for mRNA gene expression studies in platelets. Platelets were incubated for 24 h and microarray of platelet mRNA revealed that the levels of YWHAE, B2M, ITM2B, H3F3A, PF4V1 remained similar between 0 and 24 h. Further validation of the stability of these genes together with GAPDH, RN18S1, and PPIA, genes frequently used as reference genes in platelet studies, was performed using qPCR after different in vitro conditions. In addition, inter-individual stability of the genes was analyzed in diabetic patients compared with healthy matched controls. Analysis of gene stability by the software RefFinder revealed that YWHAE, PF4V1, and B2M were the most stable genes in platelets from healthy donors. In addition, YWHAE was stable between subjects. Furthermore, the potential influence of miRNA on the selected genes was investigated by knockdown of Dicer1 in the megakaryocytic cell line MEG01. YWHAE, H3F3A, B2M, and GAPDH remained unchanged over time in MEG01 cells indicating that these genes are not regulated by miRNA and hence are more stably expressed. In conclusion, YWHAE is a stable transcript in platelets and we suggest the use of YWHAE as a generic reference gene in mRNA gene expression studies.

Selection and Validation of Reference Genes for qRT-PCR in Cycas elongata.

Quantitative reverse transcription PCR (qRT-PCR) is a sensitive technique used in gene expression studies. To achieve a reliable quantification of transcripts, appropriate reference genes are required for comparison of transcripts in different samples. However, few reference genes are available for non-model taxa, and to date, reliable reference genes in Cycas elongata have not been well characterized. In this study, 13 reference genes (ACT7, TUB, UBQ, EIF4, EF1, CLATHRIN1, PP2A, RPB2, GAPC2, TIP41, MAPK, SAMDC and CYP) were chosen from the transcriptome database of C. elongata, and these genes were evaluated in 8 different organ samples. Three software programs, NormFinder, GeNorm and BestKeeper, were used to validate the stability of the potential reference genes. Results obtained from these three programs suggested that CeGAPC2 and CeRPB2 are the most stable reference genes, while CeACT7 is the least stable one among the 13 tested genes. Further confirmation of the identified reference genes was established by the relative expression of AGAMOUSE gene of C. elongata (CeAG). While our stable reference genes generated consistent expression patterns in eight tissues, we note that our results indicate that an inappropriate reference gene might cause erroneous results. Our systematic analysis for stable reference genes of C. elongata facilitates further gene expression studies and functional analyses of this species.

Defense Gene Expression Associated with Biotrophic Phase of Mycosphaerella fijiensis M. Morelet Infection in Banana.

Banana black leaf streak, caused by Mycosphaerella fijiensis M. Morelet, is a primary phytosanitary concern in export of this fruit around the world. To develop improved cultivars resistant to this disease, an understanding of host response to infection is necessary. In this study, we obtained expression data on 14,872 genes by microarray analysis in the resistant genotype Musa acuminata subsp. burmannicoides 'Calcutta 4' after inoculation with Mycosphaerella fijiensis. From these data, 16 genes were analyzed as potential reference genes and 12 genes were identified as potential early indicators of the onset of the host defense response. Subsequently, these genes were analyzed by quantitative reverse-transcription polymerase chain reaction in susceptible 'Williams' and resistant Calcutta 4. The 18S and 26S ribosomal subunit genes in both cultivars showed the best characteristics as reference genes. In all, 5 of the 12 defense genes expressed shortly after infection (peroxidase, pathogenesis-related [PR]-4, PR-10, phenylalanine ammonia-liase, and disease resistance response 1) showed overexpression in Calcutta 4 between 6 and 24 h after inoculation as opposed to Williams, which did not show overexpression after 144 h. Early induction of defense-related genes could be a key component of the resistance of the Calcutta 4 genotype against M. fijiensis. In addition, these five genes could be used as indicators of the activation of defense responses in the interaction between banana and M. fijiensis.

Selection of suitable reference genes for gene expression studies in Staphylococcus capitis during growth under erythromycin stress.

Accurate and reproducible measurement of gene transcription requires appropriate reference genes, which are stably expressed under different experimental conditions to provide normalization. Staphylococcus capitis is a human pathogen that produces biofilm under stress, such as imposed by antimicrobial agents. In this study, a set of five commonly used staphylococcal reference genes (gyrB, sodA, recA, tuf and rpoB) were systematically evaluated in two clinical isolates of Staphylococcus capitis (S. capitis subspecies urealyticus and capitis, respectively) under erythromycin stress in mid-log and stationary phases. Two public software programs (geNorm and NormFinder) and two manual calculation methods, reference residue normalization (RRN) and relative quantitative (RQ), were applied. The potential reference genes selected by the four algorithms were further validated by comparing the expression of a well-studied biofilm gene (icaA) with phenotypic biofilm formation in S. capitis under four different experimental conditions. The four methods differed considerably in their ability to predict the most suitable reference gene or gene combination for comparing icaA expression under different conditions. Under the conditions used here, the RQ method provided better selection of reference genes than the other three algorithms; however, this finding needs to be confirmed with a larger number of isolates. This study reinforces the need to assess the stability of reference genes for analysis of target gene expression under different conditions and the use of more than one algorithm in such studies. Although this work was conducted using a specific human pathogen, it emphasizes the importance of selecting suitable reference genes for accurate normalization of gene expression more generally.

Identification of Suitable Reference Genes for Gene Expression Normalization in Jatropha curcas L During Development and Under Stress Conditions Using Real Time Quantitative PCR

Jatropha curcas L represent a potential source of raw material for the production of biodiesel. The aim this study was to find potential candidate reference genes in J. curcas tissues. Three softwares were utilized to verify which would be the most stable reference genes in qPCR assay: GeNorm, NormFinder and BestKeeper. The most stable reference genes in developing J. curcas seeds suggested by GeNorm were GAPDH, UCP, actin. However, the best combinations of stable genes in each tissue were identified separately under stress conditions: EF1-α, PP2A2 and GAPDH in total stress, however, in SA stress, four genes were required for normalization: PP2A2, EF1-α, GAPDH and PUB. In PEG stress, four genes also were required: PP2A2, EF1-α, GAPDH and PUB, while in NaCl stress, five genes were necessary: PP2A2, GAPDH, EF1-α, PUB and Tβ2. These results are in accordance with two other programs used in this study (NormFinder, BestKeeper). In addition, the transcript levels of Jc-SRG-2 seem to be more correlated with stress responses than changes in transcript levels of Jc-SRG-1, mainly of leaves in exposure to 3-12 h on PEG and NaCl stress. Taken together, GAPDH and PP2A2 were regarded as being the best reference to provide guidelines for the selection of potential references genes under these study conditions.

Identification and Validation of Reference Genes for qRT-PCR Studies of Gene Expression in Dioscorea opposita.

Quantitative real-time polymerase chain reaction (qRT-PCR) is one of the most common methods for gene expression studies. Data normalization based on reference genes is essential for obtaining reliable results for qRT-PCR assays. This study evaluated potential reference genes of Chinese yam (Dioscorea opposita Thunb.), which is an important tuber crop and medicinal plant in East Asia. The expression of ten candidate reference genes across 20 samples from different organs and development stages was assessed. We identified the most stable genes for qRT-PCR studies using combined samples from different organs. Our results also suggest that different suitable reference genes or combinations of reference genes for normalization should be applied according to different organs and developmental stages. To validate the suitability of the reference genes, we evaluated the relative expression of PE2.1 and PE53, which are two genes that may be associated with microtuber formation. Our results provide the foundation for reference gene(s) selection in D. opposita and will contribute toward more accurate gene analysis studies of the genus Dioscorea.

With Reference to Reference Genes: A Systematic Review of Endogenous Controls in Gene Expression Studies.

The choice of reference genes that are stably expressed amongst treatment groups is a crucial step in real-time quantitative PCR gene expression studies. Recent guidelines have specified that a minimum of two validated reference genes should be used for normalisation. However, a quantitative review of the literature showed that the average number of reference genes used across all studies was 1.2. Thus, the vast majority of studies continue to use a single gene, with β-actin (ACTB) and/or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) being commonly selected in studies of vertebrate gene expression. Few studies (15%) tested a panel of potential reference genes for stability of expression before using them to normalise data. Amongst studies specifically testing reference gene stability, few found ACTB or GAPDH to be optimal, whereby these genes were significantly less likely to be chosen when larger panels of potential reference genes were screened. Fewer reference genes were tested for stability in non-model organisms, presumably owing to a dearth of available primers in less well characterised species. Furthermore, the experimental conditions under which real-time quantitative PCR analyses were conducted had a large influence on the choice of reference genes, whereby different studies of rat brain tissue showed different reference genes to be the most stable. These results highlight the importance of validating the choice of normalising reference genes before conducting gene expression studies.

Screening for the Most Suitable Reference Genes for Gene Expression Studies in Equine Milk Somatic Cells.

Apart from the well-known role of somatic cell count as a parameter reflecting the inflammatory status of the mammary gland, the composition of cells isolated from milk is considered as a valuable material for gene expression studies in mammals. Due to its unique composition, in recent years an increasing interest in mare's milk consumption has been observed. Thus, investigating the genetic background of horse’s milk variability presents and interesting study model. Relying on 39 milk samples collected from mares representing three breeds (Polish Primitive Horse, Polish Cold-blooded Horse, Polish Warmblood Horse) we aimed to investigate the utility of equine milk somatic cells as a source of mRNA and to screen the best reference genes for RT-qPCR using geNorm and NormFinder algorithms. The results showed that despite relatively low somatic cell counts in mare's milk, the amount and the quality of the extracted RNA are sufficient for gene expression studies. The analysis of the utility of 7 potential reference genes for RT-qPCR experiments for the normalization of equine milk somatic cells revealed some differences between the outcomes of the applied algorithms, although in both cases the KRT8 and TOP2B genes were pointed as the most stable. Analysis by geNorm showed that the combination of 4 reference genes (ACTB, GAPDH, TOP2B and KRT8) is required for apropriate RT-qPCR experiments normalization, whereas NormFinder algorithm pointed the combination of KRT8 and RPS9 genes as the most suitable. The trial study of the relative transcript abundance of the beta-casein gene with the use of various types and numbers of internal control genes confirmed once again that the selection of proper reference gene combinations is crucial for the final results of each real-time PCR experiment.

Selection and validation of reference genes for quantitative real-time PCR analysis of gene expression in Cichorium intybus.

Plant polyphenols represent a huge reservoir of bioactive compounds. Industrial chicory, an important crop from northwestern Europe, accumulates an original combination of such compounds, i.e., chlorogenic, isochlorogenic, caftaric, and chicoric acids arising from the phenylpropanoid pathway. For a complete understanding of these biochemical pathways, analyses of gene expression using quantitative real-time PCR (qRT-PCR) should be considered. Because cell cultures are a model of choice for specialized metabolism investigations, this study described for the first time the validation of reference genes for this system in chicory. Eighteen potential reference genes were obtained by mining expressed sequence tag databases of chicory for orthologs of Arabidopsis thaliana genes currently used as reference genes. Twelve genes passed the qRT-PCR standard requirements and their expression stability across different samples was tested using three distinct softwares: geNorm, NormFinder, and BestKeeper. In cell cultures grown under various conditions, TIP41 (TIP41 like protein) was shown to be the most stable gene. Further validation of the proposed reference genes was done by normalization of expression levels of a group of genes of interest. In order to assess the potentiality of the proposed list of candidate reference genes, theses genes were in parallel tested on another experimental design, i.e., chicory seedlings. In this case, the best reference gene identified was Clath (Clathrin adaptator complex subunit). The results highlight the importance of the use of properly validated reference genes to achieve relevant interpretation of qRT-PCR analyses. Here, we provide a list of reference genes suitable for future gene expression studies in chicory.