Normalization Of qRT-PCR Data Research Articles

Rifampicin alters the expression of reference genes used to normalize real-time quantitative RT-PCR data

Suitable reference genes for correct quantification of reverse transcription PCR (qRT-PCR) have to be constantly expressed in all samples under investigation. Thus, it is mandatory to determine expression stability of control genes before normalization. We aimed to establish optimum inducing concentrations for the prototypical enzyme and drug transporter inducer rifampicin in LS180 cells and concurrently assessed reference gene stability under rifampicin treatment. LS180 cells were treated with increasing concentrations of rifampicin (up to 200μM), and expression of eight different reference genes and some target genes (CYP3A4, ABCB1, and ABCC1) was quantified using real-time qRT-PCR. To check whether the results can be generalized, HepG2 cells were also investigated. We demonstrated that higher concentrations of rifampicin (>50μM) change the expression of reference genes and thus may complicate and adulterate normalization of qRT-PCR data. The results stress the need for proper validation of potential reference genes in respective cells, tissues, and particular experimental conditions. Programs like geNorm and NormFinder alone do not warrant an adequate choice of the most suitable reference gene. Scrutiny of the reference gene expression and plausibility of the data remain necessary and protect from erroneous quantification and misinterpretation of qRT-PCR data.

Validation of housekeeping genes for gene expression studies in an ice alga Chlamydomonas during freezing acclimation

Antarctic ice alga Chlamydomonas sp. ICE-L can endure extreme low temperature and high salinity stress under freezing conditions. To elucidate the molecular acclimation mechanisms using gene expression analysis, the expression stabilities of ten housekeeping genes of Chlamydomonas sp. ICE-L during freezing stress were analyzed. Some discrepancies were detected in the ranking of the candidate reference genes between geNorm and NormFinder programs, but there was substantial agreement between the groups of genes with the most and the least stable expression. RPL19 was ranked as the best candidate reference genes. Pairwise variation (V) analysis indicated the combination of two reference genes was sufficient for qRT-PCR data normalization under the experimental conditions. Considering the co-regulation between RPL19 and RPL32 (the most stable gene pairs given by geNorm program), we propose that the mean data rendered by RPL19 and GAPDH (the most stable gene pairs given by NormFinder program) be used to normalize gene expression values in Chlamydomonas sp. ICE-L more accurately. The example of FAD3 gene expression calculation demonstrated the importance of selecting an appropriate category and number of reference genes to achieve an accurate and reliable normalization of gene expression during freeze acclimation in Chlamydomonas sp. ICE-L.

Evaluation of appropriate reference genes for gene expression studies in pepper by quantitative real-time PCR

Quantitative real-time polymerase chain reaction (qRT-PCR) has been extensively used in several plant species as an accurate technique for gene expression analysis. However, the expression level of a target gene may be misconstrued due to unstable expression of the reference genes under different experimental conditions. Therefore, it is necessary to systematically evaluate these reference genes before experiments are conducted. Recently, more and more studies have focused on gene expression in pepper (Capsicum annuum L.). In this study, ten putative reference genes were chosen to identify expression stability by using geNorm and NormFinder statistical algorithms in ten different pepper sample pools, including those from different plant tissues (root, stem, leaf and flower) and from plants treated with hormones (salicylic acid and gibberellic acid) and abiotic stresses (cold, heat, salt and drought). EF1α and UEP exhibited the most stable expression across all of the tested pepper samples. For abiotic stress or different hormone treatment, the ranking of candidate reference genes was not completely consistent, except for EF1α which showed a relatively stable expression level. For different tissues, the expression of Actin1 was stable and it was considered an appropriate reference gene. It is concluded that EF1α, UEP and Actin1 are suitable reference genes for reliable qRT-PCR data normalization for the tissues and experimental conditions used in this experiment.

Selection of Reference Genes for Normalizing Quantitative Real-Time PCR Gene Expression Data with Multiple Variables in Coffea spp.

Selection and validation of appropriate reference genes should be the first step to consider in experiments based on quantitative real-time polymerase chain reaction (qRT-PCR). In this study, ten candidate genes were investigated for their stability as suitable reference genes in qRT-PCR data normalization using a diverse set of 12 Coffea cDNAs from plants from three different species/genotypes exposed to single or multiple abiotic stresses (drought and chilling, alone or in combination). Primer amplification efficiencies were calculated for all of the selected genes and varied according to each individual genotype. The expression of each gene was measured by qRT-PCR to evaluate its stability. A multiple analytical approach was followed, based on consensus merged data from four different complementary statistics, namely geNorm, BestKeeper, NormFinder, and coefficient of variation, which produced comparable but not identical results. According to this approach, the most suitable sets of reference genes for data normalization in the five experimental datasets are (1) total assay: GAPHD, Cycl, and UBQ10; (2) genotype: GAPDH, UBQ10, Ap47, and EF-1A; (3) cold stress: UBQ10, GAPDH, ACT, and EF-1A; (4) drought stress: GAPDH, ACT, EF1A, and Apt; and (5) multiple stress: UBQ10, GAPDH, ACT, and elf-4A. Normalization of gene expression using these selected genes was validated by examination of the expression of the photosynthetic-related ApoA2 gene in samples from non-stressed and stressed plants. Our results are useful to assist studies on Coffea physiology with the aim of breeding for increased tolerance to abiotic stress conditions.

Evaluation of Potential Reference Genes for Quantitative RT-PCR Analysis in Fusarium graminearum under Different Culture Conditions

Department of Medical Biotehcnology, Soonchunhyang University, Asan 336-745, Korea(Received on October 25, 2011; Accepted on November 2, 2011)The filamentous fungus Fusarium graminearum is animportant cereal pathogen. Although quantitative real-time PCR (qRT-PCR) is commonly used to analyze theexpression of important fungal genes, no detailedvalidation of reference genes for the normalization ofqRT-PCR data has been performed in this fungus.Here, we evaluated 15 candidate genes as references,including those previously described as housekeepinggenes and those selected from the whole transcriptomesequencing data. By a combination of three statisticalalgorithms (BestKeeper, geNorm, and NormFinder),the variation in the expression of these genes wasassessed under different culture conditions that favoredmycelial growth, sexual development, and trichothecenemycotoxin production. When favoring mycelial growth,GzFLO and GzUBH expression were most stable incomplete medium. Both EF1A and GzRPS16 expressionwere relatively stable under all conditions on carrotagar, including mycelial growth and the subsequentperithecial induction stage. These two genes were alsomost stable during trichothecene production. For thecombined data set, GzUBH and EF1A were selected asthe most stable. Thus, these genes are suitable referencegenes for accurate normalization of qRT-PCR data forgene expression analyses of F. graminearum and otherrelated fungi. Keywords : Fusarium graminearum, gene expression,quantitative real-time PCR, reference genesQuantitative real-time PCR (qRT-PCR) is the most sensi-tive and specific method for quantifying mRNA expressionlevels of individual target genes of interest. qRT-PCRexpression analysis has several advantages over otherconventional methods, but requires data normalization withappropriate reference gene(s) whose expression should beunaffected throughout the study conditions (Anderson etal., 2004; Bustin et al., 2004; Gutierrez et al., 2008). Severalhousekeeping genes involved in basic cellular functions,such as ribosomal genes, and those encoding actin, beta-tubulin (BTUB), translation elongation factor (EF1), andglyceraldehyde-3-phosphate dehydrogenase (GAPDH) havebeen used as reference genes for qRT-PCR in humans andother model organisms. However, several studies havedemonstrated that some of these traditional housekeepinggenes are unsuitable references because their expressionlevels vary under certain conditions (Czechowski et al.,2005; Dheda et al., 2004; Lee et al., 2002; Suzuki et al.,2000). Therefore, it is necessary to evaluate candidatereference genes for qRT-PCR expression analysis underspecific experimental conditions in the organism to bestudied. The filamentous fungus Fusarium graminearum (telo-morph: Gibberella zeae) is an important cereal pathogen,and produces mycotoxins that are harmful to humans andanimals (Desjardins and Proctor, 2007; McMullen et al.,1997). Complete genome sequencing of F. graminearum(Cuomo et al., 2007) has allowed for genome-wide expre-ssion profiling using DNA microarrays under variousdevelopmental and physiological conditions, such as my-celial growth (Guldener et al., 2006) sexual development(Hallen et al., 2007), germination (Seong et al., 2008) andpathogenesis (Guldener et al., 2006; Stephens et al., 2008).Although the microarray data in these studies were notvalidated by qRT-PCR, it has become the most commonmethod to obtain specific expression patterns of genesidentified from gene expression profiles (Chen et al., 2011;Liu et al., 2011; Pandolfi et al., 2010) or functional studies(Lee et al., 2009; Lysoe et al., 2009). However, theexpression stabilities of the housekeeping genes used asreferences for qRT-PCR analysis in even these studies havenot been properly validated. The objective of this study was to identify F. gramine-arum reference genes suitable for normalization of qRT-PCR data under different conditions such as mycelialgrowth, sexual development, and mycotoxin production.We selected 15 candidate references, including the commonhousekeeping genes used in previous studies (Chen et al.,2011; Lee et al., 2009; Liu et al., 2011; Lysoe et al., 2009;Pandolfi et al., 2010) and a new set of genes selected from

Rpb2is a reliable reference gene for quantitative gene expression analysis in the dermatophyteTrichophyton rubrum

The selection of reference genes used for data normalization to quantify gene expression by real-time PCR amplifications (qRT-PCR) is crucial for the accuracy of this technique. In spite of this, little information regarding such genes for qRT-PCR is available for gene expression analyses in pathogenic fungi. Thus, we investigated the suitability of eight candidate reference genes in isolates of the human dermatophyte Trichophyton rubrum subjected to several environmental challenges, such as drug exposure, interaction with human nail and skin, and heat stress. The stability of these genes was determined by geNorm, NormFinder and Best-Keeper programs. The gene with the most stable expression in the majority of the conditions tested was rpb2 (DNA-dependent RNA polymerase II), which was validated in three T. rubrum strains. Moreover, the combination of rpb2 and chs1 (chitin synthase) genes provided for the most reliable qRT-PCR data normalization in T. rubrum under a broad range of biological conditions. To the best of our knowledge this is the first report on the selection of reference genes for qRT-PCR data normalization in dermatophytes and the results of these studies should permit further analysis of gene expression under several experimental conditions, with improved accuracy and reliability.

Selection and use of reference genes in mouse mammary glands

Obtaining quantitative data concerning gene expression is important for understanding milk synthesis in mammary glands. Quantitative real-time PCR (qRT-PCR) is an efficient tool to calculate gene expression; however, it is necessary to find valid reference genes for normalization of qRT-PCR data. We applied the geNorm software to eight commonly used reference genes to identify the most stable and optimal genes for the mouse mammary gland. Based on this analysis, HPRT, RPL and GAPDH are the most appropriate reference genes for data normalization. We tested the expression of the alpha-lactalbumin and fatty acid synthase genes using these three reference genes, both normalized and non-normalized. The normalized mRNA expression ratio was significantly different from the non-normalized ratio. We recommend the use of these three reference genes for the normalization of qRT-PCR data in gene expression studies of mouse mammary glands.

Predator-induced defences in Daphnia pulex: Selection and evaluation of internal reference genes for gene expression studies with real-time PCR

BackgroundThe planktonic microcrustacean Daphnia pulex is among the best-studied animals in ecological, toxicological and evolutionary research. One aspect that has sustained interest in the study system is the ability of D. pulex to develop inducible defence structures when exposed to predators, such as the phantom midge larvae Chaoborus. The available draft genome sequence for D. pulex is accelerating research to identify genes that confer plastic phenotypes that are regularly cued by environmental stimuli. Yet for quantifying gene expression levels, no experimentally validated set of internal control genes exists for the accurate normalization of qRT-PCR data.ResultsIn this study, we tested six candidate reference genes for normalizing transcription levels of D. pulex genes; alpha tubulin (aTub), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), TATA box binding protein (Tbp) syntaxin 16 (Stx16), X-box binding protein 1 (Xbp1) and CAPON, a protein associated with the neuronal nitric oxide synthase, were selected on the basis of an earlier study and from microarray studies. One additional gene, a matrix metalloproteinase (MMP), was tested to validate its transcriptional response to Chaoborus, which was earlier observed in a microarray study. The transcription profiles of these seven genes were assessed by qRT-PCR from RNA of juvenile D. pulex that showed induced defences in comparison to untreated control animals. We tested the individual suitability of genes for expression normalization using the programs geNorm, NormFinder and BestKeeper. Intriguingly, Xbp1, Tbp, CAPON and Stx16 were selected as ideal reference genes. Analyses on the relative expression level using the software REST showed that both classical housekeeping candidate genes (aTub and GAPDH) were significantly downregulated, whereas the MMP gene was shown to be significantly upregulated, as predicted. aTub is a particularly ill suited reference gene because five copies are found in the D. pulex genome sequence. When applying aTub for expression normalization Xbp1 and Tbp are falsely reported as significantly upregulated.ConclusionsOur results suggest that the genes Xbp1, Tbp, CAPON and Stx16 are suitable reference genes for accurate normalization in qRT-PCR studies using Chaoborus-induced D. pulex specimens. Furthermore, our study underscores the importance of verifying the expression stability of putative reference genes for normalization of expression levels.

Validation of housekeeping genes for quantitative real-time PCR in in-vivo and in-vitro models of cerebral ischaemia

BackgroundStudies of gene expression in experimental cerebral ischaemia models can contribute to understanding the pathophysiology of brain ischaemia and to identifying prognostic markers and potential therapeutic targets. The normalization of relative qRT-PCR data using a suitable reference gene is a crucial prerequisite for obtaining reliable conclusions. No validated housekeeping genes have been reported for the relative quantification of the mRNA expression profile activated in in-vitro ischaemic conditions, whereas for the in-vivo model different reference genes have been used.The present study aims to determine the expression stability of ten housekeeping genes (Gapdh, β2m, Hprt, Ppia, Rpl13a, Oaz1, 18S rRNA, Gusb, Ywhaz and Sdha) to establish their suitability as control genes for in-vitro and in-vivo cerebral ischaemia models.ResultsThe expression stability of the candidate reference genes was evaluated using the 2-ΔC'T method and ANOVA followed by Dunnett's test. For the in-vitro model using primary cultures of rat astrocytes, all genes analysed except for Rpl13a and Sdha were found to have significantly different levels of mRNA expression. These different levels were also found in the case of the in-vivo model of pMCAO in rats except for Hprt, Sdha and Ywhaz mRNA, where the expression did not vary. Sdha and Ywhaz were identified by geNorm and NormFinder as the two most stable genes.ConclusionWe have validated endogenous control genes for qRT-PCR analysis of gene expression in in-vitro and in-vivo cerebral ischaemia models. For normalization purposes, Rpl13a and Sdha are found to be the most suitable genes for the in-vitro model and Sdha and Ywhaz for the in-vivo model. Genes previously used as housekeeping genes for the in-vivo model in the literature were not validated as good control genes in the present study, showing the need for careful evaluation for each new experimental setup.

Selection of REFERENCE Genes for Normalization of qRT-PCR Data Derived From FFPE Breast Tumors

Gene expression analysis of formalin-fixed paraffin-embedded (FFPE) material by quantitative real-time polymerase chain reaction is a valuable tool for the retrospective analysis of clinical samples. The degraded nature of RNA from FFPE tissue can lead to variation in detection of gene expression between samples, hence, it is necessary to select reference genes that can reflect this variation. To identify suitable references for the normalization of target genes in FFPE breast tumors, expression of 10 potential references were measured in 10 endocrine therapy-naive, primary invasive breast tumors. The most stably expressed reference genes were identified with the use of the freely available algorithm geNorm. Using the geometric mean of MRPL19, TBP, and TFRC as a reference factor for FFPE tissue, there was a good correlation of ERalpha and HER2 measurements between FFPE and matched frozen tumors, indicating that this combination of reference genes successfully removed the effect of RNA degradation on quantitative real-time polymerase chain reaction analysis.

Normalization of qRT-PCR data: the necessity of adopting a systematic, experimental conditions-specific, validation of references

Quantitative RT-PCR (reverse transcription polymerase chain reaction, also known as qRT-PCR or real-time RT-PCR) has been used in large proportions of transcriptome analyses published to date. The accuracy of the results obtained by this method strongly depends on accurate transcript normalization using stably expressed genes, known as references. Statistical algorithms have been developed recently to help validate reference genes but, surprisingly, this robust approach is under-utilized in plants. Instead, putative 'housekeeping' genes tend to be used as references without any proper validation. The concept of normalization in transcript quantification is introduced here and the factors affecting its reliability in qRT-PCR are discussed in an attempt to convince molecular biologists, and non-specialists, that systematic validation of reference genes is essential for producing accurate, reliable data in qRT-PCR analyses, and thus should be an integral component of them.

Identification of Endogenous Reference Genes for qRT-PCR Analysis in Normal Matched Breast Tumor Tissues

Quantitative gene expression measurements from tumor tissue are frequently compared with matched normal and/or adjacent tumor tissue expression for diagnostic marker gene selection as well as assessment of the degree of transcriptional deregulation in cancer. Selection of an appropriate reference gene (RG) or an RG panel, which varies depending on cancer type, molecular subtypes, and the normal tissues used for interindividual calibration, is crucial for the accurate quantification of gene expression. Several RG panels have been suggested in breast cancer for making comparisons among tumor subtypes, cell lines, and benign/malignant tumors. In this study, expression patterns of 15 widely used endogenous RGs (ACTB, TBP, GAPDH, SDHA, HPRT, HMBS, B2M, PPIA, GUSB, YWHAZ2, PGK1, RPLP0, PUM1, MRPL19, and RPL41), and three candidate genes that were selected through analysis of two independent microarray datasets (IL22RA1, TC22, ZNF224) were determined in 23 primary breast tumors and their matched normal tissues using qRT-PCR. Additionally, 18S rRNA, ACTB, and SDHA were tested using randomly primed cDNAs from 13 breast tumor pairs to assess the rRNA/mRNA ratio. The tumors exhibited significantly lower rRNA/mRNA ratio when compared to their normals, on average. The expression of the studied RGs in breast tumors did not exhibit differences in terms of grade, ER, or PR status. The stability of RGs was examined based on two different statistical models, namely GeNorm and NormFinder. Among the 18 tested endogenous reference genes, ACTB and SDHA were identified as the most suitable reference genes for the normalization of qRT-PCR data in the analysis of normal matched tumor breast tissue pairs by both programs. In addition, the expression of the gelsolin (GSN) gene, a well-known downregulated target in breast tumors, was analyzed using the two most suitable genes and different RG combinations to validate their effectiveness as a normalization factor (NF). The GSN expression of the tumors used in this study was significantly lower than that of normals showing the effectivity of using ACTB and SDHA as suitable RGs in this set of tumor-normal tissue panel. The combinational use of the best performing two RGs (ACTB and SDHA) as a normalization factor can be recommended to minimize sample variability and to increase the accuracy and resolution of gene expression normalization in tumor-normal paired breast cancer qRT-PCR studies.

Normalization of microRNA expression levels in quantitative RT-PCR assays: Identification of suitable reference RNA targets in normal and cancerous human solid tissues

Proper normalization is a critical but often an underappreciated aspect of quantitative gene expression analysis. This study describes the identification and characterization of appropriate reference RNA targets for the normalization of microRNA (miRNA) quantitative RT-PCR data. miRNA microarray data from dozens of normal and disease human tissues revealed ubiquitous and stably expressed normalization candidates for evaluation by qRT-PCR. miR-191 and miR-103, among others, were found to be highly consistent in their expression across 13 normal tissues and five pair of distinct tumor/normal adjacent tissues. These miRNAs were statistically superior to the most commonly used reference RNAs used in miRNA qRT-PCR experiments, such as 5S rRNA, U6 snRNA, or total RNA. The most stable normalizers were also highly conserved across flash-frozen and formalin-fixed paraffin-embedded lung cancer tumor/NAT sample sets, resulting in the confirmation of one well-documented oncomir (let-7a), as well as the identification of novel oncomirs. These findings constitute the first report describing the rigorous normalization of miRNA qRT-PCR data and have important implications for proper experimental design and accurate data interpretation.

P4 Genes for normalization of qRT-PCR data in breast cancer

P4 Genes for normalization of qRT-PCR data in breast cancer

Selection of reference genes for quantitative RT-PCR studies in striped dolphin (Stenella coeruleoalba) skin biopsies

BackgroundOdontocete cetaceans occupy the top position of the marine food-web and are particularly sensitive to the bioaccumulation of lipophilic contaminants. The effects of environmental pollution on these species are highly debated and various ecotoxicological studies have addressed the impact of xenobiotic compounds on marine mammals, raising conservational concerns. Despite its sensitivity, quantitative real-time PCR (qRT-PCR) has never been used to quantify gene induction caused by exposure of cetaceans to contaminants. A limitation for the application of qRT-PCR is the need for appropriate reference genes which allow the correct quantification of gene expression. A systematic evaluation of potential reference genes in cetacean skin biopsies is presented, in order to validate future qRT-PCR studies aiming at using the expression of selected genes as non-lethal biomarkers.ResultsTen commonly used housekeeping genes (HKGs) were partially sequenced in the striped dolphin (Stenella coeruleoalba) and, for each gene, PCR primer pairs were specifically designed and tested in qRT-PCR assays. The expression of these potential control genes was examined in 30 striped dolphin skin biopsy samples, obtained from specimens sampled in the north-western Mediterranean Sea. The stability of selected control genes was determined using three different specific VBA applets (geNorm, NormFinder and BestKeeper) which produce highly comparable results. Glyceraldehyde-3P-dehydrogenase (GAPDH) and tyrosine 3-monooxygenase (YWHAZ) always rank as the two most stably expressed HKGs according to the analysis with geNorm and Normfinder, and are defined as optimal control genes by BestKepeer. Ribosomal protein L4 (RPL4) and S18 (RPS18) also exhibit a remarkable stability of their expression levels. On the other hand, transferrin receptor (TFRC), phosphoglycerate kinase 1 (PGK1), hypoxanthine ribosyltransferase (HPRT1) and β-2-microglobin (B2M) show variable expression among the studied samples and appear as less suitable reference genes for data normalization.ConclusionIn this work, we have provided essential background information for the selection of control genes in qRT-PCR studies of cetacean skin biopsies, as a molecular technique to investigate ecotoxicological hazard in marine mammals. Of 10 HKGs tested, those encoding for YWHAZ and GAPDH appear as the most reliable control genes for the normalization of qRT-PCR data in the analysis of striped dolphin skin biopsies. Potentially useful reference genes are also those encoding for ribosomal proteins L4 and S18.