Reference Genes For Normalization Research Articles

Selection of reference genes for measuring the expression of aiiO in Ochrobactrum quorumnocens A44 using RT-qPCR

Reverse transcription quantitative PCR (RT-qPCR), a method of choice for quantification of gene expression changes, requires stably expressed reference genes for normalization of data. So far, no reference genes were established for the Alphaproteobacteria of the genus Ochrobactrum. Here, we determined reference genes for gene expression studies in O. quorumnocens A44. Strain A44 was cultured under 10 different conditions and the stability of expression of 11 candidate genes was evaluated using geNorm, NormFinder and BestKeeper. Most stably expressed genes were found to be rho, gyrB and rpoD. Our results can facilitate the choice of reference genes in the related Ochrobactrum strains. O. quorumnocens A44 is able to inactivate a broad spectrum of N-acyl homoserine lactones (AHLs) – the quorum sensing molecules of many Gram-negative bacteria. This activity is attributed to AiiO hydrolase, yet it remains unclear whether AHLs are the primary substrate of this enzyme. Using the established RT-qPCR setup, we found that the expression of the aiiO gene upon exposure to two AHLs, C6-HLS and 3OC12-HSL, does not change above the 1-fold significance threshold. The implications of this finding are discussed in the light of the role of quorum sensing-interfering enzymes in the host strains.

Selection of suitable reference genes for qRT-PCR analysis of Begonia semperflorens under stress conditions.

Begonia semperflorens (B. semperflorens), belonging to the family Begoniaceae, has now been widely cultivated worldwide and is famous for its ornamental plants with colourful flowers and distinctive leaves. The selection of appropriate internal reference genes is very important to accurately determine target gene expression via quantitative real-time PCR. However, internal reference gene selection has never been conducted in B. semperflorens. In this study, seven candidate reference genes of B. semperflorens, including 18S ribosomal RNA (Bs18S), pentatricopeptide repeat-containing protein (BsPPR), actin-related protein 5 isoform X2 (BsACT), DNAJ homologue subfamily C member 17 (BsDNAJ), glyceraldehyde-3-phosphate dehydrogenase (BsGAPDH), NAD-dependent malic enzyme 59kDa isoform, mitochondria (BsNAD-ME), and peptidyl-prolyl cis-trans isomerase CYP26-2, chloroplast (BsCYP), which were obtained from our previous studies, were selected. The stabilities of these genes under stress conditions were analysed using geNorm and NormFinder. Validation of target gene expressions, including phenylalanine ammonia-lyase (BsPAL) and respiratory burst oxidase homologue D (BsRBOHD) under biotic and abiotic conditions, phenylalanine ammonia-lyase (BsPAL), anthocyanidin synthase (BsANS), chalcone synthase (BsCHS), and flavanone-3-hydroxylase (BsF3H) under low temperature, using these seven internal reference genes for normalisation further confirmed the stabilities of the selected genes and indicated the need for reference gene selection for normalising gene expressions in B. semperflorens. Of the seven candidate reference genes, the combination of BsACT, BsDNAJ, and BsNAD-ME was the ideal reference gene set for normalising gene expression in samples under biotic conditions. BsCYP combined with BsACT or BsGAPDH was the best reference gene pair under abiotic conditions. BsACT and BsPPR could be combined to normalise gene expression under low temperature. Our results will benefit future studies on gene expression in plants of Begoniaceae.

Identification of suitable reference genes for normalization of RT-qPCR data in eggplant fruit and shoot borer (Leucinodes orbonalis Guenée)

The eggplant shoot and fruit borer, Leucinodes orbonalis Guenee (Lepidoptera: Crambidae) is a monophagous and destructive insect pest of eggplant. L. orbonalis developed multiple insecticide resistance that led to frequent field control failures in many countries. The possible reversal of resistance in L. orbonalis requires an understanding of the expression dynamics of individual genes conferring resistance to insecticides through gene expression analysis. The availability of reference genes (RG) to normalize transcript data of target genes, achieved by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), is mandatory. Hence, the present study investigated the stable expression pattern of nine putative reference genes viz., actin, 28S ribosomal protein S18c, mitochondrial isoform X2 (28SM), calnexin, β-tubulin, 28S ribosomal protein S3 mitochondrial (28SR3), TATA box binding protein (TATA), elongation factor-1α (EF-1α), glyceraldehyde-3-phosphate-dehydrogenase (GAPDH), and ubiquitin 60S ribosomal protein (Ubiquitin) to select the most stable genes that could be used as reference genes for target transcript gene normalization. Different algorithms such as ΔCt, geNorm, NormFinder and BestKeeper were used to assess gene expression stability. By integrating these data into online RefFinder tool, 28SR3 and GAPDH, were chosen as highly suitable RGs based on their stable expression pattern.

Identification of stably expressed genes for normalization of gene expression data in oral tumors: a preliminary analysis

We sought to identify stably expressed genes in tumors of gingivo-buccal region and tongue from untreated as well as treated patients. The study was undertaken in view of the ambiguity with regards to the choice of reference genes for normalization of gene expression data from gingivo-buccal region and tongue. This aspect was also examined in tumors from treated patients since it could provide clues for such analyses in the assessment of treatment modalities in the future. Expression of ten candidate housekeeping genes, identified in array-based studies, was tested using TaqMan based semi-quantitative real-time PCR. Thirty-five buccal mucosa derived (18 from treated patients) and 15 tongue tumors (8 from treated patients) were studied. Most stable genes were identified based on the consensus between the results of the three methods, Comparative δCt, BestKeeper and NormFinder, used for data analysis. CHMP2A and VPS29 were identified as the most stably expressed genes suitable for normalization of data from buccal-mucosa tumors, whereas RPS13 and PSMB2 were indicated for similar specimens from treated patients. The same criteria identified stable expression of PSMB2 and PUM1 in tumors from tongue and OAZ1 and RPS13 for the post-treatment tongue tumors. We have identified stably expressed genes in common oral cancers which can be used for normalization of the gene expression data. Results also established differences in tumors arising at different sites of the oral cavity and highlighted further changes following exposure to therapy.

Genome-wide identification of internal reference genes for normalization of gene expression values during endosperm development in wheat.

Internal reference genes that are stably expressed are essential for normalization in comparative expression analyses. However, gene expression varies significantly among species, organisms, tissues, developmental stages, stresses, and treatments. Therefore, identification of stably expressed reference genes in developmental endosperm of bread wheat is important for expression analysis of endosperm genes. As the first study to systematically screen for reference genes across different developmental stages of wheat endosperm, nine genes were selected from among 76 relatively stable genes based on high-throughput RNA sequencing data. The expression stability of these candidate genes and five traditional reference genes was assessed by real-time quantitative PCR combined with three independent algorithms: geNorm, NormFinder, and BestKeeper. The results showed that ATG8d was the most stable gene during wheat endosperm development, followed by Ta54227, while the housekeeping gene GAPDH, commonly used as an internal reference, was the least stable. ATG8d and Ta54227 together formed the optimal combination of reference genes. Comparative expression analysis of glutenin genes indicated that credible quantification could be achieved by normalization against ATG8d in developmental endosperm. The stably expressed gene characterized here can act as a proper internal reference for expression analysis of wheat endosperm genes, especially nutrient- and nutrient synthesis-related genes.

Identification of reliable reference genes for quantitative real-time PCR normalization in pitaya

BackgroundA suitable reference gene is an important prerequisite for guarantying accurate and reliable results in quantitative real-time PCR (qRT-PCR) analyses. However, there is no absolute universality in reference genes among different species. It’s hard to find an ideal reference gene to fit for different tissues and growth periods. Pitaya (Hylocereus) is commercially produced as a new fruit crop at a large scale in tropical and subtropical regions. To date, there is no report on the identification of the most reliable reference genes for qRT-PCR normalization in pitaya.ResultsIn this study, six candidate reference genes i.e. Actin(1), GAPDH, UBC(1), UBC(2) EF1-α(1) and histone(1) were selected from thirty-nine typical candidate reference genes to determine the most stable reference genes for qRT-PCR normalization in different tissues, temperature stresses and fruit developmental stages of pitaya. Among the six candidate reference genes, Actin(1) and EF1-α(1) were the most stable gene according to calculations of three statistical methods (GeNorm, NormFinder and BestKeeper) while UBC(1) and UBC(2) showed the lowest expression stability. The six candidate reference genes were further validated by comparing expression profiles of key genes related to betalain biosynthesis at flesh coloration stages of Guanhuahong (Hylocereus monacanthus) and Guanhuabai (H. undatus) pitayas. Actin(1) was recommended the best reference gene for accurate normalization of qRT-PCR data.ConclusionsIn this study, the stability of the selected reference genes for normalizing the qRT-PCR data were identified from pitaya. Actin(1) was the most stably expressed genes in different tissues and fruit developmental stages in pitaya. The present work provides the first data of reference gene identification for pitaya and will facilitate further studies in molecular biology and gene function on Hylocereus and other closely related species.

Selection of Reference Genes for Normalization of Gene Expression Data in Blood of Machado-Joseph Disease/Spinocerebellar Ataxia Type 3 (MJD/SCA3) Subjects.

Alongside with the emergent clinical trials for Machado-Joseph disease/Spinocerebellar ataxia type 3 (MJD/SCA3) comes the need to identify molecular biomarkers of disease that can be tracked throughout the trial. MJD is an autosomal dominant neurodegenerative disorder caused by expansion of a CAG repeat in the coding region of the ATXN3 gene. Previous findings indicate the potential of transcriptional alterations in blood of MJD patients as biomarkers of disease. Accurate quantification of gene expression levels by quantitative real-time PCR (qPCR) depends on data normalization, usually performed using reference genes. Because the expression level of routinely used housekeeping genes may vary in multiple biological and experimental conditions, reference gene controls should be validated in each specific experimental design. Here, we aimed to evaluate the expression behavior of five housekeeping genes previously reported as stably expressed in peripheral blood of patients from several disorders-peptidylprolyl isomerase B (PPIB), TNF receptor associated protein 1 (TRAP1), beta-2-microglobulin (B2M), 2,4-dienoyl-CoA reductase 1 (DECR1), and folylpolyglutamate synthase (FPGS). Expression levels of these five genes were assessed by qPCR in blood from MJD subjects (preataxic and patients) and matched controls. While all housekeeping genes, here studied, were stably expressed in our sets of samples, TRAP1 showed to be the most stable gene by NormFinder and BestKeeper. We, therefore, conclude that any of these genes could be used as reference gene in future qPCR studies using blood samples from MJD subjects.

Selection of reference genes for flowering pathway analysis in the masting plants, Celmisia lyallii and Chionochloa pallens, under variable environmental conditions

Mast flowering is characterised by mass synchronised flowering at irregular intervals over a wide geographical area. An understanding of the molecular drivers of mast flowering requires expression analysis of key developmentally regulated gene(s). Reverse transcription-quantitative PCR is the gold standard technique used to assess expression of target gene(s) and to validate high-throughput sequencing data. Selection and validation of appropriate reference gene(s), used as normalisation factors in transcript abundance analysis, is an essential step to avoid ambiguous expression results. Eight candidate reference genes were assessed to select the best internal normalisation factors in naturally growing masting plants Chionochloa pallens and Celmisia lyallii. Statistical packages geNorm, Normfinder, BestKeeper, ΔCt and RefFinder were used to determine the expression stability in plants translocated to different altitudes and sampled across the season. GAPDH and PP2a in Celmisia and ExP and THP in Chionochloa were found to be the best pairs of reference genes for normalisation of the gene expression data. Our study revealed environmentally-induced changes in reference gene expression, information that will be utilised as we investigate flowering phenology of masting plants under global climatic change.

Reference genes for real-time RT-PCR expression studies in an Antarctic Pseudomonas exposed to different temperature conditions.

Psychrophilic and psychrotolerant bacteria from permanently cold environments may be the most abundant extremophiles on Earth and yet little is known on how they cope with temperature stress. Real-time reverse transcription PCR (RT-qPCR) is a powerful technique that could shed light on this matter but it requires pre-validated reference genes for normalization of data to get accurate results. In this study, we assessed the expression stability of eight candidate genes for the psychrotolerant Antarctic isolate Pseudomonas sp. AU10 during exponential growth under 4°C and 30°C, and after a cold-shock. Using the software programs BestKeeper and geNorm we validated recA, ftsZ, 16S rRNA, and rpoD as reference genes and we suggested the combination of recA and ftsZ for qPCR data normalization. Our results provide a starting point for gene expression studies in Antarctic Pseudomonas concerning temperature-related physiology and also for the validation of reference genes in other cold-adapted bacterial species.

Selection of suitable candidate genes for miRNA expression normalization in Yellow River Carp (Cyprinus carpio. var)

Yellow River carp is widely cultivated in the world due to its economic value in aquaculture, and the faster growth of females compared to males. It is believed that microRNAs (miRNA) are involved in gonadal differentiation and development. qPCR is the most preferred method for miRNA functional analysis. Reliable reference genes for normalization in qRT-PCR are the key to ensuring the accuracy of this method. The aim of present research was to evaluate as well as identify the efficacy of reference genes for miRNA expression using qRT-PCR in Yellow River carp. Nine ncRNAs (miR-101, miR-23a, let7a, miR-26a, miR-146a, miR-451, U6, 5S, and 18S) were chosen and tested in four sample sets: (1) different tissues in adult carp, (2) different tissues in juvenile carp, (3) different early developmental stages of carp, and (4) different developmental stages of carp gonads. The stability and suitability values were calculated using NormFinder, geNorm, and BestKeeper software. The results showed that 5S was a suitable reference gene in different tissues of adult and juvenile carp. The genes 5S, 18S, and U6 were the most stable reference genes in the early developmental stages of carp. Let-7a and miR-23a were considered as the suitable reference genes in the development of gonads. All these reference genes were subsequently validated using miR-430. The results showed that genes 5S and 18S were the most suitable reference genes to normalize miRNA expression under normal growth conditions in early different developmental stages. The genes Let-7a, and miR-23a were the most suitable in different developmental stages. The present study is the first comprehensive study of the stability of miRNA reference genes in Yellow River carp, providing valuable as well as basic data for investigating more accurate miRNA expression during gonadal differentiation and development of carp.

Screening for quantitative real-time PCR reference genes with high stable expression using the mRNA-sequencing data for pear

Housekeeping genes were considered to have a constant expression without spatio-temporal difference. They are commonly used as the internal reference for PCR-based gene expression analysis. However, increasing evidence revealed that the expression of housekeeping genes also varied with tissues and treatments. Previous studies for reference gene identification were usually limited to a small number of housekeeping genes based on their expression stability comparison. In this study, reference genes for pear fruit were initially screened on the transcriptome level with 10 messenger RNA (mRNA)-seq datasets from the fruit of two pear species across important developmental stages. Ten potential reference genes were chosen for validation using the mRNA-seq data from fruit skin libraries. Five potential reference genes are proposed that can be used as suitable controls for PCR-based expression studies in pear fruit. Four of the selected genes had homologous annotation of important roles in the basic biological processes. Sulfhydryl oxidase 2 (SOX2) and protein phosphatase (PP) 2A also showed high stable expression in both pear fruit and other tissues and should be more widely applicable as reference genes in different tissues or species. This study provided a comprehensive view for transcriptional analysis in pear fruit, which will help researchers to select reference genes for normalization against target genes by qRT-PCR across different developmental stages of the fruit as well as for different tissues and species.

Validation of Reference Genes for Gene Expression Normalization in RAW264.7 Cells under Different Conditions.

RAW264.7 is a macrophage strain derived from mice tumour and shows a significant ability in antigen uptake. Real-time quantitative PCR (RT-qPCR) is one of the most commonly used methods in gene studies and requires suitable reference genes to normalize and quantitate the expression of gene of interest with sensitivity and specificity. However, suitable reference genes in RAW264.7 cells have not yet been identified for accurate gene expression quantification. In the current study, we evaluated expression levels of ten candidate reference genes in RAW264.7 cells under different conditions. RT-qPCR results indicated significant differences in the expression levels among the ten reference genes. Statistical analyses were carried out using geNorm, NormFinder, and BestKeeper software to further investigate the stability of the reference genes. Integrating the results from the three analytical methods, cytochrome c-1 and hydroxymethylbilane synthase were found to be the most stable and therefore more suitable reference genes, while ribosomal protein L4 and cyclophilin A were the least stable. This study emphasises the importance of identifying and selecting the most stable reference genes for normalization and provides a basis for future gene expression studies using RAW264.7 cells.

Comparative transcriptomics-based selection of suitable reference genes for normalization of RT-qPCR experiments in drought-stressed leaves of three European Quercus species

Reference genes for normalization of reverse transcription quantitative real-time PCR (RT-qPCR) experiments were selected and evaluated for drought-stressed leaves of three European Quercus species (Q. ilex, Q. pubescens, and Q. robur). A drought experiment was conducted over the course of 2 years. In the first year, a comparative transcriptome analysis was conducted between control and drought-stressed plants in all three species. Based on this analysis, six genes which showed a low fold change and low individual variation of normalized expression values were selected as novel candidate reference genes. The six novel candidate genes were homologs of the serine/threonineprotein kinase At1g54610 (At1g54610), ATP synthase gamma chain (ATPC1), far-red elongated hypocotyl 3-like (FHY3), 50S ribosomal protein L13 (RPL13), serine/threonine-protein phosphatise PP1 (TOPP2), and splicing factor U2af small subunit B-like (U2AF35B). As a control, the two classical candidate reference genes glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and elongation factor 1 alpha (EF1a) were included. Cross-species primers were designed for all candidate reference genes. The reference genes were evaluated in samples from three dates in the second year of the drought experiment. Four different algorithms (Bestkeeper, the comparative Ct method, geNorm, and Normfinder) were utilized to analyze expression stability. The results of the algorithms were summarized, and the most stable genes and optimal number of reference genes were identified for every species. These results demonstrated that, in each species, at least four of the novel reference genes are more stably expressed than the classical reference genes and that two reference genes are the optimal number for each species. One novel reference gene, At1g54610, was found to be among the two most stable reference genes across all three species; the second most stable gene was found to be TOPP2 in Q. ilex, U2AF35B in Q. pubescens, and FHY3 in Q. robur. Reference genes are crucial to normalize qPCR data and to identify stress-responsive genes, e.g., in the context of a search for drought-tolerant Quercus genotypes able to withstand the predicted increase of drought events in large parts of Europe during climate change.

Reference genes selection for Calotropis procera under different salt stress conditions.

Calotropis procera is a perennial Asian shrub with significant adaptation to adverse climate conditions and poor soils. Given its increased salt and drought stress tolerance, C. procera stands out as a powerful candidate to provide alternative genetic resources for biotechnological approaches. The qPCR (real-time quantitative polymerase chain reaction), widely recognized among the most accurate methods for quantifying gene expression, demands suitable reference genes (RGs) to avoid over- or underestimations of the relative expression and incorrect interpretation. This study aimed at evaluating the stability of ten RGs for normalization of gene expression of root and leaf of C. procera under different salt stress conditions and different collection times. The selected RGs were used on expression analysis of three target genes. Three independent experiments were carried out in greenhouse with young plants: i) Leaf100 = leaf samples collected 30 min, 2 h, 8 h and 45 days after NaCl-stress (100 mM NaCl); ii) Root50 and iii) Root200 = root samples collected 30 min, 2 h, 8 h and 1day after NaCl-stress (50 and 200 mM NaCl, respectively). Stability rank among the three algorithms used showed high agreement for the four most stable RGs. The four most stable RGs showed high congruence among all combination of collection time, for each software studied, with minor disagreements. CYP23 was the best RG (rank of top four) for all experimental conditions (Leaf100, Root50, and Root200). Using appropriated RGs, we validated the relative expression level of three differentially expressed target genes (NAC78, CNBL4, and ND1) in Leaf100 and Root200 samples. This study provides the first selection of stable reference genes for C. procera under salinity. Our results emphasize the need for caution when evaluating the stability RGs under different amplitude of variable factors.

Reference gene selection for qRT-PCR in Brazilian-ginseng [Pfaffia glomerata (Spreng.) Pedersen] as affected by various abiotic factors

The determination of the best reference gene is essential to improve and guarantee the accuracy of the qPCR technique. Thus, the objective of this work was to evaluate the normalization genes efficiency for qRT-PCR studies of Pfaffia glomerata, a species with marked medicinal and industrial interests due to the production of the phytoecdysteroid 20-hydroxyecdysone (20-E). We have selected four candidates as reference genes in P. glomerata: elongation factor-1α (EF-1α), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), spectrin-like (SPT) and α-tubulin (TUA), and tested their expression stabilities as affected by abiotic factors (salt stress, drought stress, irradiance, photoperiod and CO2 enrichment), using the following methods: NormFinder, geNorm, BestKeeper, ΔCt and RefFinder. Also, the Phantom gene, which belongs to the 20-E biosynthesis pathway, was targeted to validate the most stable reference gene for each abiotic factor. The GAPDH was the most stable gene under all assessed abiotic factors, as well as the most stable when data from all experiments were taken together, which was confirmed by all the software used. The use of more or less stable reference genes for normalization significantly changes the interpretation of the qPCR data, evidencing the importance of choosing the most appropriate housekeeping gene for each expression assay. Based on our results, we recommend GAPDH to be used for normalization of qPCR expression data in P. glomerata in diverse abiotic conditions. This work is the first report on the validation of reference genes in P. glomerata and will be fundamental for further gene expression studies in this important medicinal species. GAPDH is the most stable gene to be used for the normalization in qPCR analyzes in [Pfaffia glomerata (Spreng.) Pedersen] under the different abiotic factors.

Selection and validation of reference genes for quantitative expression analysis of miRNAs and mRNAs in Poplar

BackgroundQuantitative reverse transcriptase polymerase chain reaction (qRT-PCR) is a rapid and sensitive approach to identify miRNA and protein-coding gene expression in plants. However, because of the specially designated reverse transcription and shorter PCR products, very few reference genes have been identified for the quantitative analysis of miRNA expression in plants, and different internal reference genes are needed to normalize the expression of miRNAs and mRNA genes respectively. Therefore, it is particularly important to select the suitable common reference genes for normalization of quantitative PCR of miRNA and mRNA.ResultsIn this study, a modified reverse transcription PCR protocol was adopted for selecting and validating universal internal reference genes of mRNAs and miRNAs. Eight commonly used reference genes, four stably expressed novel genes in Populus tremula, three small noncoding RNAs and three conserved miRNAs were selected as candidate genes, and the stability of their expression was examined across a set of 38 tissue samples from four developmental stages of poplar clone 84K (Populus alba × Populus glandulosa). The expression stability of these candidate genes was evaluated systematically by four algorithms: geNorm, NormFinder, Bestkeeper and DeltaCt. The results showed that Eukaryotic initiation factor 4A III (EIF4A) and U6-2 were suitable for samples of the callus stage; U6-1 and U6-2 were best for the seedling stage; Protein phosphatase 2A-2 (PP2A-2) and U6-1 were best for the plant stage; and Protein phosphatase 2A-2 (PP2A-2) and Oligouridylate binding protein 1B (UBP) were the best reference genes in the adventitious root (AR) regeneration stage.ConclusionsThe purpose of this study was to identify the most appropriate reference genes for qRT-PCR of miRNAs and mRNAs in different tissues at several developmental stages in poplar. U6-1, EIF4A and PP2A-2 were the three most appropriate reference genes for qRT-PCR normalization of miRNAs and mRNAs during the plant regeneration process, and PP2A-2 and UBP represent the best reference genes in the AR regeneration stage of poplar. This work will benefit future studies of expression and function analysis of miRNAs and their target genes in poplar.

Identification of suitable reference genes for gene expression studies in rat skeletal muscle following sciatic nerve crush injury.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is a molecular biological method used to assess gene expression characterized by high simplicity, effectiveness, specificity and sensitivity. The selection of a suitable reference gene for normalization is critical for the accuracy of quantitative results. Peripheral nerve injury is a common clinical disorder that affects multiple tissues and organs, including peripheral nerves, neurons and the innervated muscles. Numerous genes are differentially expressed in skeletal muscles during muscle denervation and reinnervation following peripheral nerve injury. The identification of a suitable reference gene in innervated muscles following nerve injury may improve the understanding of the alterations in gene expression in the processes of peripheral nerve repair and regeneration. Therefore, in the present study, by using a rat sciatic nerve crush model, the expression levels of various housekeeping genes were examined. In particular, the expression levels of 13 housekeeping genes, including 18S ribosomal RNA, actin β, ankyrin repeat domain 27, cyclophilin A, GAPDH, hypoxanthine phosphoribosyltransferase 1 (HPRT1), mitochondrial ribosomal protein L10, phosphoglycerate kinase 1, RPTOR independent companion of mammalian target of rapamycin complex 2, TATA-box binding protein, ubiquitin C, UBX domain protein 11 and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein ζ, were investigated in gastrocnemius muscles. The geNorm and NormFinder analyses suggested that the expression level of HPRT1 was particularly stable in gastrocnemius muscles following rat sciatic nerve crush injury. Therefore, HPRT1 may be used as a reference gene for the normalization of gene expression data generated by RT-qPCR.

Selection of reference genes for the quantitative real-time PCR normalization of gene expression in Isatis indigotica fortune

BackgroundIsatis indigotica, a traditional Chinese medicine, produces a variety of active ingredients. However, little is known about the key genes and corresponding expression profiling involved in the biosynthesis pathways of these ingredients. Quantitative real-time polymerase chain reaction (qRT-PCR) is a powerful, commonly-used method for gene expression analysis, but the accuracy of the quantitative data produced depends on the appropriate selection of reference genes.ResultsIn this study, the systematic analysis of the reference genes was performed for quantitative real-Time PCR normalization in I. indigotica. We selected nine candidate reference genes, including six traditional housekeeping genes (ACT, α-TUB, β-TUB, UBC, CYP, and EF1-α), and three newly stable internal control genes (MUB, TIP41, and RPL) from a transcriptome dataset of I. indigotica, and evaluated their expression stabilities in different tissues (root, stem, leaf, and petiole) and leaves exposed to three abiotic treatments (low-nitrogen, ABA, and MeJA) using geNorm, NormFinder, BestKeeper, and comprehensive RefFind algorithms. The results demonstrated that MUB and EF1-α were the two most stable reference genes for all samples. TIP41 as the optimal reference gene for low-nitrogen stress and MeJA treatment, while ACT had the highest ranking for ABA treatment and CYP was the most suitable for different tissues.ConclusionsThe results revealed that the selection and validation of appropriate reference genes for normalizing data is mandatory to acquire accurate quantification results. The necessity of specific internal control for specific conditions was also emphasized. Furthermore, this work will provide valuable information to enhance further research in gene function and molecular biology on I. indigotica and other related species.

Reliable reference genes for expression analysis of proliferating and adipogenically differentiating human adipose stromal cells

BackgroundThe proliferation and adipogenic differentiation of adipose stromal cells (ASCs) are complex processes comprising major phenotypical alterations driven by up- and downregulation of hundreds of genes. Quantitative RT-PCR can be employed to measure relative changes in the expression of a gene of interest. This approach requires constitutively expressed reference genes for normalization to counteract inter-sample variations due to differences in RNA quality and quantity. Thus, a careful validation of quantitative RT-PCR reference genes is needed to accurately measure fluctuations in the expression of genes. Here, we evaluated candidate reference genes applicable for quantitative RT-PCR analysis of gene expression during proliferation and adipogenesis of human ASCs with the immunophenotype DLK1+/CD34+/CD90+/CD105+/CD45−/CD31−.MethodsWe evaluated the applicability of 10 candidate reference genes (GAPDH, TBP, RPS18, EF1A, TFRC, GUSB, PSMD5, CCNA2, LMNA and MRPL19) using NormFinder, geNorm and BestKeeper software.ResultsThe results indicate that EF1A and MRPL19 are the most reliable reference genes for quantitative RT-PCR analysis of proliferating ASCs. PSMD5 serves as the most reliable endogenous control in adipogenesis. CCNA2 and LMNA were among the least consistent genes.ConclusionsApplying these findings for future gene expression analyses will help elucidate ASC biology.

Selection of suitable reference genes for quantitative real-time PCR gene expression analysis in Mulberry (Morus alba L.) under different abiotic stresses.

Mulberry (Morus alba L.) is the sole food source for the mulberry silkworm, Bombyx mori and therefore important for sericulture industry. Different abiotic stress conditions like drought, salt, heat and cold stress adversely affect the productivity and quality of mulberry leaves. Quantitative real time PCR (qPCR) is a reliable and widely used method to identify abiotic stress responsive genes and molecular mechanism in different plant species. Selection of suitable reference genes is important requirement for normalizing the expression of genes through qRT-PCR study. In the present study, we have selected eight candidate reference genes in mulberry for analyzing their expression stability in different abiotic stress treatments including drought, salt, heat and cold stresses. The expression stability of these reference genes was determined using geNorm, NormFinder and RefFinder statistical algorithms. The results showed that Ubiquitin and protein phosphatase 2A regulatory subunit A (PP2A) were the most stable genes across all the treatment samples. However, analysis of individual stresses revealed different expression profiles and stability of reference genes. Actin3 and PP2A were most stable in drought and salt conditions respectively. RPL3 most preferred in heat stress and Ubiquitin was most stable in cold stress. We propose the ubiquitin and PP2A are the preferred reference genes for normalization of gene expression data from abiotic stresses. In addition, Actin3 are preferred for drought stress, PP2A for salt stress, RPL3 for heat stress and Ubiquitin for cold stress studies.