Selection Of Appropriate Reference Genes Research Articles

Validation of Reference Genes for Quantitative Real-Time PCR in Bovine PBMCs Transformed and Non-transformed by Theileria annulata.

Theileria annulata is a tick-borne intracellular protozoan parasite that causes tropical theileriosis, a fatal bovine lymphoproliferative disease. The parasite predominantly invades bovine B lymphocytes and macrophages and induces host cell transformation by a mechanism that is not fully comprehended. Analysis of signaling pathways by quantitative real-time PCR (qPCR) could be a highly efficient means to understand this transformation mechanism. However, accurate analysis of qPCR data relies on selection of appropriate reference genes for normalization, yet few papers on T. annulata contain evidence of reference gene validation. We therefore used the geNorm and NormFinder programs to evaluate the stability of 5 candidate reference genes; 18S rRNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ACTB (β-actin), PRKG1 (protein kinase cGMP-dependent, type I) and TATA box binding protein (TBP). The results showed that 18S rRNA was the reference gene most stably expressed in bovine PBMCs transformed and non-transformed with T. annulata, followed by GAPDH and TBP. While 18S rRNA and GAPDH were the best combination, these 2 genes were chosen as references to study signaling pathways involved in the transformation mechanism of T. annulata.

The importance of selecting the appropriate reference genes for quantitative real time PCR as illustrated using colon cancer cells and tissue

Quantitative real-time reverse-transcription polymerase chain reaction (RT-qPCR) remains the most sensitive technique for nucleic acid quantification. Its popularity is reflected in the remarkable number of publications reporting RT-qPCR data. Careful normalisation within RT-qPCR studies is imperative to ensure accurate quantification of mRNA levels. This is commonly achieved through the use of reference genes as an internal control to normalise the mRNA levels between different samples. The selection of appropriate reference genes can be a challenge as transcript levels vary with physiology, pathology and development, making the information within the transcriptome flexible and variable. In this study, we examined the variation in expression of a panel of nine candidate reference genes in HCT116 and HT29 2-dimensional and 3-dimensional cultures, as well as in normal and cancerous colon tissue. Using normfinder we identified the top three most stable genes for all conditions. Further to this we compared the change in expression of a selection of PKC coding genes when the data was normalised to one reference gene and three reference genes. Here we demonstrated that there is a variation in the fold changes obtained dependent on the number of reference genes used. As well as this, we highlight important considerations namely; assay efficiency tests, inhibition tests and RNA assessment which should also be implemented into all RT-qPCR studies. All this data combined demonstrates the need for careful experimental design in RT-qPCR studies to help eliminate false interpretation and reporting of results.

Selection of reliable reference genes in eutopic and ectopic endometrium for quantitative expression studies

Physiological changes during menstrual cycle cause the endometrium and endometriosis to develop specific kind of tissues, especially in regard to the gene expression profiles, which may include also housekeeping genes, commonly used as reference genes (RGs) in quantitative studies. Reverse transcription, followed by quantitative polymerase chain reaction (RT-qPCR) is the most precise and commonly used method in gene expression studies. In order to reduce effects of technical approaches and biological variability of gene's expression level, the studies often employ RGs in experimental data normalization. However, the expression of RGs is not always stable and depends on several variables. Thus, the selection of appropriate RG is one of the most significant steps to obtain reliable results in RT-qPCR-based methods. With the usage of RT-qPCR, we researched the expression of seven genes (ACTB, B2M, G6PD, GAPD, GUSB, HPRT and PPIA) as reliable reference genes in eutopic and ectopic endometrial tissue specimens obtained during standard surgery of women of reproductive age. Stability of expression level was analyzed by the most universal MS Excel plug-ins including: geNorm, NormFinder and BestKeeper. The descriptive statistics were evaluated using Statistica software. The distribution of threshold (Ct) values was not equal. We identified genes with higher expression level (referring to Ct values) such as ACTB and B2M, medium e.g., GAPD and low expression level, e.g., G6PD and HPRT. We demonstrated that the stability of the analyzed reference genes was not homogenous, and different algorithms pointed to PPIA, GAPD and B2M as the most stable ones in eutopic and ectopic endometrium. On the contrary to these, GUSB and G6PD were the most unstable ones. In RT-qPCR-based analyses of gene expression level in eutopic and ectopic endometrium, we strongly recommend that a minimum of two reference genes are to be used and we determined that the most suitable seem to be PPIA and GAPD.

Pathogenesis-related genes as tools for discovering the response of onion defence system against Iris yellow spot virus infection

The Iris yellow spot virus (IYSV) is a viral pathogen of onions and causes severe damage and economic loss in affected onion crops. Pathogenesis-related (PR) genes are part of the innate immune response that onions harbour against viral diseases, which to date remains unclear. Using a sensitive and reliable real-time quantitative PCR method, the dynamic expression of five different genes in infected onions were investigated after biological inoculation with virulent isolate of Thrips tabaci (Lindeman). The transcription levels of PR1, PR2, PR3, PR4, and PR5 genes were highly expressed 1 day post inoculation (dpi). Furthermore, statistical analysis revealed a significant change in peak expression levels of PR1 after 8 dpi and PR3 after 9 dpi. In contrast, the expression level change for the other genes was only moderate. Further, we determined and ranked the expression stability of three reference genes (EF1-α, 18S rRNA, and β-actin) using geNorm and NormFinder. The overall analysis demonstrated that β-actin is the most informative gene, which can be utilised as an internal control for quantitative gene expression. Our study findings not only provide guidelines for selection of appropriate reference genes in virally infected onions, but also valuable information concerning immune response related genes associated with the initial plant defences against viral infection. Moreover, the PR1 gene appears to be a disease-specific gene related to the IYSV infection in onion tissues.

β2-Microglobulin is an appropriate reference gene for RT-PCR-based gene expression analysis of hematopoietic stem cells.

Real-time reverse transcription polymerase chain reaction (RT-PCR) is regarded as one of the most useful and powerful tools for characterizing hematopoietic stem cells (HSCs), because samples of extremely small cell numbers can be analyzed. The expression levels determined by RT-PCR are based on relative quantification; therefore, the selection of an appropriate reference gene with a relatively stable expression level under most conditions is crucial. Here, we determined that beta2-microglobulin (B2m) is an appropriate reference gene for analyzing mouse HSCs by a novel method using single-cell RT-PCR. Clonally sorted HSCs were subjected to RT reactions with exogenous RNA fragments and then to real-time PCR. Next, the relative gene expression levels of 4 well-known housekeeping genes were quantified in each single cell sample based on the threshold cycle of exogenous RNA. The analysis revealed that B2m expression was reproducibly detected in almost all HSCs and that B2m had the most stable expression level among the compared genes, even after the cells had been cultured under various conditions. Thus, our results indicate that B2m can reliably be used as a reference gene for the relative quantification of expression levels in HSCs across various conditions. Furthermore, our work proposes a novel method for the selection of appropriate reference genes.

Evaluation and Selection of Appropriate Reference Genes for Real-Time Quantitative PCR Analysis of Gene Expression in Nile Tilapia (Oreochromis niloticus) during Vaccination and Infection.

qPCR as a powerful and attractive methodology has been widely applied to aquaculture researches for gene expression analyses. However, the suitable reference selection is critical for normalizing target genes expression in qPCR. In the present study, six commonly used endogenous controls were selected as candidate reference genes to evaluate and analyze their expression levels, stabilities and normalization to immune-related gene IgM expression during vaccination and infection in spleen of tilapia with RefFinder and GeNorm programs. The results showed that all of these candidate reference genes exhibited transcriptional variations to some extent at different periods. Among them, EF1A was the most stable reference with RefFinder, followed by 18S rRNA, ACTB, UBCE, TUBA and GAPDH respectively and the optimal number of reference genes for IgM normalization under different experiment sets was two with GeNorm. Meanwhile, combination the Cq (quantification cycle) value and the recommended comprehensive ranking of reference genes, EF1A and ACTB, the two optimal reference genes, were used together as reference genes for accurate analysis of immune-related gene expression during vaccination and infection in Nile tilapia with qPCR. Moreover, the highest IgM expression level was at two weeks post-vaccination when normalized to EF1A, 18S rRNA, ACTB, and EF1A together with ACTB compared to one week post-vaccination before normalizing, which was also consistent with the IgM antibody titers detection by ELISA.

Identification of reference genes for quantitative expression analysis of microRNAs and mRNAs in barley under various stress conditions.

For accurate and reliable gene expression analysis using quantitative real-time reverse transcription PCR (qPCR), the selection of appropriate reference genes as an internal control for normalization is crucial. We hypothesized that non-coding, small nucleolar RNAs (snoRNAs) would be stably expressed in different barley varieties and under different experimental treatments, in different tissues and at different developmental stages of plant growth and therefore might prove to be suitable reference genes for expression analysis of both microRNAs (miRNAs) and mRNAs. In this study, we examined the expression stability of ten candidate reference genes in six barley genotypes under five experimental stresses, drought, fungal infection, boron toxicity, nutrient deficiency and salinity. We compared four commonly used housekeeping genes; Actin (ACT), alpha-Tubulin (α-TUB), Glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ADP-ribosylation factor 1-like protein (ADP), four snoRNAs; (U18, U61, snoR14 and snoR23) and two microRNAs (miR168, miR159) as candidate reference genes. We found that ADP, snoR14 and snoR23 were ranked as the best of these candidates across diverse samples. For accurate and reliable gene expression analysis using quantitative real-time reverse transcription PCR (qPCR), the selection of appropriate reference genes as an internal control for normalization is crucial. We hypothesized that non-coding, small nucleolar RNAs (snoRNAs) would be stably expressed in different barley varieties and under different experimental treatments, in different tissues and at different developmental stages of plant growth and therefore might prove to be suitable reference genes for expression analysis of both microRNAs (miRNAs) and mRNAs. In this study, we examined the expression stability of ten candidate reference genes in six barley genotypes under five experimental stresses, drought, fungal infection, boron toxicity, nutrient deficiency and salinity. We compared four commonly used housekeeping genes; Actin (ACT), alpha-Tubulin (α-TUB), Glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ADP-ribosylation factor 1-like protein (ADP), four snoRNAs; (U18, U61, snoR14 and snoR23) and two microRNAs (miR168, miR159) as candidate reference genes. We found that ADP, snoR14 and snoR23 were ranked as the best of these candidates across diverse samples. Additionally, we found that miR168 was a suitable reference gene for expression analysis in barley. Finally, we validated the performance of our stable and unstable candidate reference genes for both mRNA and miRNA qPCR data normalization under different stress conditions and demonstrated the superiority of the stable candidates. Our data demonstrate the suitability of barley snoRNAs and miRNAs as potential reference genes for miRNA and mRNA qPCR data normalization under different stress treatments.

Validation of suitable reference genes for gene expression analysis in the halophyte Salicornia europaea by real-time quantitative PCR.

Real-time quantitative polymerase chain reaction (RT-qPCR), a reliable technique for quantifying gene expression, requires stable reference genes to normalize its data. Salicornia europaea, a stem succulent halophyte with remarkable salt resistance and high capacity for ion accumulation, has not been investigated with regards to the selection of appropriate reference genes for RT-qPCR. In this study, the expression of 11 candidate reference genes, GAPDH (Glyceraldehyde 3-phosphate dehydrogenase), Actin, α-Tub (α-tubulin), β-Tub (β-tubulin), EF1-α (Elongation factor 1-α), UBC (Ubiquitin-conjugating enzyme), UBQ (Polyubiquitin), CYP (Cyclophilin), TIP41 (TIP41-like protein), CAC (Clathrin adaptor complexes), and DNAJ (DnaJ-like protein), was analyzed in S. europaea samples, which were classified into groups according to various abiotic stresses (NaCl, nitrogen, drought, cold and heat), tissues and ages. Three commonly used software programs (geNorm, NormFinder and BestKeeper) were applied to evaluate the stability of gene expression, and comprehensive ranks of stability were generated by aggregate analysis. The results show that the relatively stable genes for each group are the following: (1) CAC and UBC for whole samples; (2) CAC and UBC for NaCl stress; (3) Actin and α-Tub for nitrogen treatment; (4) Actin and GAPDH for drought stress; (5) α-Tub and UBC for cold stress; (6) TIP41 and DNAJ for heat stress; (7) UBC and UBQ for different tissues; and (8) UBC and Actin for various developmental stages. These genes were validated by comparing transcriptome profiles. Using two stable reference genes was recommended in the normalization of RT-qPCR data. This study identifies optimal reference genes for RT-qPCR in S. europaea, which will benefit gene expression analysis under these conditions.

Selection of appropriate reference genes for RT-qPCR analysis in Berkshire, Duroc, Landrace, and Yorkshire pigs

Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is the most reliable molecular biology technique for assessment of mRNA expression levels. However, to obtain the accurate RT-qPCR results, the expression levels of genes of interest should be normalized with appropriate reference genes and optimal numbers of reference genes. In this study, we assessed the expression stability of 15 well-known candidate reference genes (ACTB, ALDOA, B2M, GAPDH, HPAR1, HSPCB, PGK1, POLR2G, PPIA, RPL4, RPS18, SDHA, TBP, TOP2B, and YWHAZ) in seven body tissues (liver, lung, kidney, spleen, stomach, small intestine, and large intestine) of Berkshire, Landrace, Duroc, and Yorkshire pigs using three excel-based programs, geNorm, NormFinder, and BestKeeper. Combination analysis of these three programs showed that the stable and appropriate reference genes are PPIA, TBP, and HSPCB in Berkshire pigs; PPIA, TBP, RPL4, and RPS18 in Landrace pigs; PPIA and TBP in Duroc pigs; and PPIA, TOP2B, RPL4, and RPS18 in Yorkshire pigs. Because the four pig breeds had different suitable reference genes, the selection of appropriate reference genes is essential in RT-qPCR analyses. Taken together, our data could help to select reliable reference genes for the normalization of expression levels of various target genes in pigs.

Selection of appropriate reference genes in eggplant for quantitative gene expression studies under different experimental conditions

Quantitative real-time polymerase chain reaction (RT-qPCR) has become one of the most widely used methods for gene expression analysis, and the evaluation of appropriate reference genes should be the first step to consider in experiments based on RT-qPCR. In this work, nine candidate reference genes, including 18sRNA, CYP, UBQ, GAPDH, TUB, TUA, EF1, ACTIN and RPOB, were investigated for their expression stability in five different eggplant sample pools using three algorithms: geNorm, NormFinder and RefFinder. The samples were collected under different experimental conditions, including those from different plant tissues, hormone treatments, biotic and abiotic stresses. Our results demonstrated that the expression stability varied from different experimental conditions between reference genes. In general, GAPDH and 18sRNA had a good performance under most experimental conditions, whereas TUB and RPOB exhibited poor stability. This study should provide guidelines for the selection of reference genes for gene expression studies in eggplant.

Evaluation of reference genes for quantitative real-time PCR in oil palm elite planting materials propagated by tissue culture.

BackgroundThe somatic embryogenesis tissue culture process has been utilized to propagate high yielding oil palm. Due to the low callogenesis and embryogenesis rates, molecular studies were initiated to identify genes regulating the process, and their expression levels are usually quantified using reverse transcription quantitative real-time PCR (RT-qPCR). With the recent release of oil palm genome sequences, it is crucial to establish a proper strategy for gene analysis using RT-qPCR. Selection of the most suitable reference genes should be performed for accurate quantification of gene expression levels.ResultsIn this study, eight candidate reference genes selected from cDNA microarray study and literature review were evaluated comprehensively across 26 tissue culture samples using RT-qPCR. These samples were collected from two tissue culture lines and media treatments, which consisted of leaf explants cultures, callus and embryoids from consecutive developmental stages. Three statistical algorithms (geNorm, NormFinder and BestKeeper) confirmed that the expression stability of novel reference genes (pOP-EA01332, PD00380 and PD00569) outperformed classical housekeeping genes (GAPDH, NAD5, TUBULIN, UBIQUITIN and ACTIN). PD00380 and PD00569 were identified as the most stably expressed genes in total samples, MA2 and MA8 tissue culture lines. Their applicability to validate the expression profiles of a putative ethylene-responsive transcription factor 3-like gene demonstrated the importance of using the geometric mean of two genes for normalization.ConclusionsSystematic selection of the most stably expressed reference genes for RT-qPCR was established in oil palm tissue culture samples. PD00380 and PD00569 were selected for accurate and reliable normalization of gene expression data from RT-qPCR. These data will be valuable to the research associated with the tissue culture process. Also, the method described here will facilitate the selection of appropriate reference genes in other oil palm tissues and in the expression profiling of genes relating to yield, biotic and abiotic stresses.

Evaluation of internal control for gene expression in Phalaenopsis by quantitative real-time PCR.

The selection of appropriate reference genes is one of the most important steps to obtain reliable results for normalizing quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) of MADS-box gene in Phalaenopsis. In this study, we cloned 12 candidate reference genes including 18S ribosomal RNA (18S), elongation factor 1 alpha (EF1α), cytoskeletal structural protein actin (ACT1, ACT2, ACT3, ACT4, ACT5), ubiquitin protein (UBQ1 and UBQ2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and the cytoskeletal structural proteins α-tubulin (TUA) and β-tubulin (TUB) in Phalaenopsis and evaluated their expression reliability. The expression of these candidate reference genes was analyzed using geNorm and normFinder software packages; the results showed that ACT2 and ACT4 were the highest stability reference genes for all experiment sets based on normFinder, followed by ACT1 or ACT3, while ACT3 and ACT4 were the highest stability reference genes for most experiment sets based on geNorm, then TUB or others. Taken together, Actin genes were the higher stability reference genes for all tissues at total developmental stages, and similar results came from analysis by normFinder. According to geNorm analysis, ACT3 and ACT4 were the most stable reference genes for all tissues tested and tissues at reproductive stages; TUB and ACT5 or ACT4 were the most stable reference genes for vegetative tissues or roots. The most stable reference genes for all vegetative tissues and only leaves were ACT4 and ACT5, ACT2 and ACT3, respectively; ACT1 and ACT3 were the most stable genes and sufficient for reliable normalization of flower tissues. While EF1α, UBQ1, UBQ2, and GAPDH were found to be unsuitable as a reference gene in our analysis for flower tissues, total tissues, and reproductive stages; UBQ2 and 18S were identified as the least stable reference genes for vegetative tissues at different stages, different tissues at vegetative stages; TUA and 18S were the least reliable reference genes for the samples from roots at all developmental stages. This is the first systematic report on the selection of reference genes in Phalaenopsis, and these data will facilitate future work on gene expression in orchid.

Evaluation of reference genes for quantitative real-time PCR in the brain, pituitary, and gonads of songbirds

Quantitative real-time PCR (qPCR) is becoming a popular tool for the quantification of gene expression in the brain and endocrine tissues of songbirds. Accurate analysis of qPCR data relies on the selection of appropriate reference genes for normalization, yet few papers on songbirds contain evidence of reference gene validation. Here, we evaluated the expression of ten potential reference genes (18S, ACTB, GAPDH, HMBS, HPRT, PPIA, RPL4, RPL32, TFRC, and UBC) in brain, pituitary, ovary, and testis in two species of songbirds: zebra finch and white-throated sparrow. We used two algorithms, geNorm and NormFinder, to assess the stability of these reference genes in our samples. We found that the suitability of some of the most popular reference genes for target gene normalization in mammals, such as 18S, depended highly on tissue type. Thus, they are not the best choices for brain and gonad in these songbirds. In contrast, we identified alternative genes, such as HPRT, RPL4 and PPIA, that were highly stable in brain, pituitary, and gonad in these species. Our results suggest that the validation of reference genes in mammals does not necessarily extrapolate to other taxonomic groups. For researchers wishing to identify and evaluate suitable reference genes for qPCR in songbirds, our results should serve as a starting point and should help increase the power and utility of songbird models in behavioral neuroendocrinology.

Reference Gene Selection for Gene Expression Studies Using Quantitative Real-Time PCR Normalization in Atropa belladonna

Quantitative PCR (qPCR) is a powerful tool for measuring gene expression levels. Accurate and reproducible results are dependent on the correct choice of reference genes for data normalization. Atropa belladonna is a commercial plant species from which pharmaceutical tropane alkaloids are extracted. In this study, eight candidate reference genes, namely 18S ribosomal RNA (18S), actin (ACT), cyclophilin (CYC), elongation factor 1α (EF-1α), β-fructosidase (FRU), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK), and beta-tubulin (TUB), were selected and their expression stabilities studied to determine their suitability for normalizing gene expression in A. belladonna. The expression stabilities of these genes were analyzed in the root, stem, and leaf under cold, heat, NaCl, UV-B, methyl jasmonate, salicylic acid, and abscisic acid treatments using geNorm, NormFinder, and BestKeeper. The statistical algorithms indicated that PGK was a reliable gene for normalizing gene expression under most of the experimental conditions. The pairwise value analysis showed that two genes were sufficient for proper expression normalization, except when analyzing gene expression in heat-treated roots. However, the choice of the second reference gene depended on specific conditions. Finally, the relative expression level of the PMT gene of A. belladonna was detected to validate the selection of PGK a reliable reference gene. In summary, our results should guide the selection of appropriate reference genes for gene expression studies in A. belladonna under different organs and abiotic stress conditions.

Technical note: Selecting the best references in gene expression experiments in liver of cows receiving glucogenic supplements during the transition period

Measuring gene expression is a commonly used method to monitor the reaction of cells and tissues to changing nutritional or physiological conditions. Selection of appropriate reference genes is a crucial point in gene expression experiments using real-time PCR techniques. Expression of the “ideal” reference gene should not be affected by the experimental treatments or physiological state of the tissue, organ, or the whole organism. Many programs are available from which to choose the most stable reference gene. In this study, 4 algorithms—ΔCt, BestKeeper, NormFinder, and geNorm—were used to assess the expression stability of 5 candidate reference genes: β-actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein S9 (RPS9), ribosomal protein L32 (RPL32), and TATA-box-binding protein (TBP), for use in an experiment aimed at measuring gene expression in the liver of cows fed glucogenic supplements in the transition from pregnancy to lactation. The results demonstrated that RPS9 and RPL32 were the most stably expressed in the liver under the conditions of the present experiment; the least stably expressed was ACTB.

Defining reference genes for qPCR normalization to study biotic and abiotic stress responses in Vigna mungo

Expression of ACT, EF1A; H2A, EF1A, ACTand 18S, TUB showed stability under MYMIV, salinity and drought stress, respectively; these are recommended as reference genes for qPCR normalization in Vigna mungo. Accurate gene expression profiling through qPCR depends on selection of appropriate reference gene(s) for normalization. Due to lack of unanimous internal standard, suitable constitutively expressed reference genes are selected that exhibit stable expression under diverse experimental conditions. In this communication, a comparative evaluation of stability among seven V. mungo genes encoding actin (ACT), histone H2A (H2A), elongation factor 1-alpha (EF1A), 18S rRNA (18S), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), cyclophilin (CYP) and tubulin (TUB) under biotic (MYMIV) and abiotic (drought and salinity) stress conditions has been attempted. Specificity and amplification efficiency for each primer pair were verified; however, cumulative assessment of their accumulated transcripts revealed no uniformity. Therefore, individual stability and suitability of these seven candidates have been assessed in silico, by two widely used algorithms, geNorm and Normfinder. Based on the computed results, high stability was obtained for ACT and EF1A during MYMIV stress, while H2A, EFIA and ACT were found to be most suitable in salinity stress experiments and TUB and 18S during drought treatments. Combinations of ACT/TUB or ACT/EFIA were recommended for their use in the pooled analysis, while expression of 18S and CYP showed greater variations and therefore considered unsuitable as reference genes. Additionally, precise quantification of the target gene VmPRX under these stresses was shown to be a function of reference genes' stability, which tends to get affected when normalized with the least stable genes. Hence, use of these normalizers will facilitate accurate and reliable analyses of gene expression in V. mungo.

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.

Importance of Suitable Reference Gene Selection for Quantitative RT-PCR during ATDC5 Cells Chondrocyte Differentiation

Real-time quantitative reverse transcription-polymerase chain reaction (qPCR) is an efficient and accurate method to detect and compare patterns of gene expression. The reliability of qPCR is highly dependent on the selection of appropriate reference genes used for normalization. By analyzing 16 potential candidates of reference genes (GAPDH, Actb, 18 s, PGK1, Hprt, Tbp, Rpl5, B2M, Gusb, Ppia, UBC, Sdha, Eef1a1, H2afz, Tkt and Ldha) through geNorm, we identified Ppia, Tbp, Hprt and Eef1a1 as the most stable reference genes while UBC, B2M, Gusb as the least stable ones during the chondrocyte differentiation of ATDC5 cells. Considering the low expression of Eef1a1 and Tbp would cause divergent results for they failed to provide accurate normalization for RNA extraction and reverse transcription efficiency, we recommended the use of Ppia and Hprt as the most suitable genes to normalize qPCR. In addition, although GAPDH, Actb and 18 s were usually adopted in most of studies using ATDC5 cells, they were found unstable and then were not ideal reference genes for qPCR assay in ATDC5 cells chondrocyte differentiation. Also, we further confirmed that the Ppia and Hprt worked well during chondrocyte differentiation of mouse mesenchymal cells.

Selection of New Appropriate Reference Genes for RT-qPCR Analysis via Transcriptome Sequencing of Cynomolgus Monkeys (Macaca fascicularis)

In the investigation of the expression levels of target genes, reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is the most accurate and widely used method. However, a normalization step is a prerequisite to obtain accurate quantification results from RT-qPCR data. Therefore, many studies regarding the selection of reference genes have been carried out. Recently, these studies have involved large-scale gene analysis methods such as microarray and next generation sequencing. In our previous studies, we analyzed large amounts of transcriptome data from the cynomolgus monkey. Using a modification of this large-scale transcriptome sequencing dataset, we selected and compared 12 novel candidate reference genes (ARFGAP2, ARL1, BMI1, CASC3, DDX3X, MRFAP1, ORMDL1, RSL24D1, SAR1A, USP22, ZC3H11A, and ZRANB2) and 4 traditionally used reference genes (ACTB, GAPDH, RPS19, and YWHAZ) in 13 different whole-body tissues by the 3 well-known programs geNorm, NormFinder, and BestKeeper. Combined analysis by these 3 programs showed that ADP-ribosylation factor GTPase activating protein 2 (ARFGAP2), morf4 family associated protein 1 (MRFAP1), and ADP-ribosylation factor-like 1 (ARL1) are the most appropriate reference genes for accurate normalization. Interestingly, 4 traditionally used reference genes were the least stably expressed in this study. For this reason, selection of appropriate reference genes is vitally important, and large-scale analysis is a good method for finding new candidate reference genes. Our results could provide reliable reference gene lists for future studies on the expression of various target genes in the cynomolgus monkey.