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Evaluation of Candidate Reference Genes for Gene Expression Normalization in Brassica juncea Using Real Time Quantitative RT-PCR.
PLoS One. 2012;7(5):e36918
Authors: Chandna R, Augustine R, Bisht NC
Abstract
The real time quantitative reverse transcription PCR (qRT-PCR) is becoming increasingly important to gain insight into function of genes. Given the increased sensitivity, ease and reproducibility of qRT-PCR, the requirement of suitable reference genes for normalization has become important and stringent. It is now known that the expression of internal control genes in living organism vary considerably during developmental stages and under different experimental conditions. For economically important Brassica crops, only a couple of reference genes are reported till date. In this study, expression stability of 12 candidate reference genes including ACT2, ELFA, GAPDH, TUA, UBQ9 (traditional housekeeping genes), ACP, CAC, SNF, TIPS-41, TMD, TSB and ZNF (new candidate reference genes), in a diverse set of 49 tissue samples representing different developmental stages, stress and hormone treated conditions and cultivars of Brassica juncea has been validated. For the normalization of vegetative stages the ELFA, ACT2, CAC and TIPS-41 combination would be appropriate whereas TIPS-41 along with CAC would be suitable for normalization of reproductive stages. A combination of GAPDH, TUA, TIPS-41 and CAC were identified as the most suitable reference genes for total developmental stages. In various stress and hormone treated samples, UBQ9 and TIPS-41 had the most stable expression. Across five cultivars of B. juncea, the expression of CAC and TIPS-41 did not vary significantly and were identified as the most stably expressed reference genes. This study provides comprehensive information that the new reference genes selected herein performed better than the traditional housekeeping genes. The selection of most suitable reference genes depends on the experimental conditions, and is tissue and cultivar-specific. Further, to attain accuracy in the results more than one reference genes are necessary for normalization.
PMID: 22606308 [PubMed - as supplied by publisher]
Identification of stable endogenous control genes for transcriptional profiling of photon, proton and carbon-ion irradiated cells.
Radiat Oncol. 2012 May 17;7(1):70
Authors: Sharungbam GD, Schwager C, Chiblak S, Brons S, Hlatky L, Haberer T, Debus J, Abdollahi A
Abstract
ABSTRACT: BACKGROUND: Quantitative analysis of transcriptional regulation of genes is a prerequisite for a better understanding of the molecular mechanisms of action of different radiation qualities such as photon, proton or carbon ion irradiation. Microarrays and real-time quantitative RT-PCR (qRT-PCR) are considered the two cornerstones of gene expression analysis. In interpreting these results it is critical to normalize the expression levels of the target genes by that of appropriately selected endogenous control genes (ECGs) or housekeeping genes. We sought to systematically investigate common ECG candidates for their stability after different radiation modalities in different human cell lines by qRT-PCR. We aimed to identify the most robust set of ECGs or housekeeping genes for transcriptional analysis in irradiation studies. METHODS: We tested the expression stability of 32 ECGs in three human cancer cell lines. The epidermoid carcinoma cells (A431), the non small cell lung carcinoma cells (A549) and the pancreatic adenocarincoma cells (BxPC3) were irradiated with photon, proton and carbon ions. Expression Heat maps, clustering and statistic algorithms were employed using SUMO software package. The expression stability was evaluated by computing: mean, standard deviation, ANOVA, coefficient of variation and the stability measure (M) given by the geNorm algorithm. RESULTS: Expression analysis revealed significant cell type specific regulation of 18 out of 32 ECGs (p < 0.05). A549 and A431 cells shared a similar pattern of ECG expression as the function of different radiation qualities as compared to BxPC3. Of note, the ribosomal protein 18S, one of the most frequently used ECG, was differentially regulated as the function of different radiation qualities (p [less than or equal to] 0.01). A comprehensive search for the most stable ECGs using the geNorm algorithm identified 3 ECGs for A431 and BxPC3 to be sufficient for normalization. In contrast, 6 ECGs were required to properly normalize expression data in the more variable A549 cells. Considering both variables tested, i.e. cell type and radiation qualities, 5 genes-- RPLP0, UBC, PPIA, TBP and PSMC4-- were identified as the consensus set of stable ECGs. CONCLUSIONS: Caution is warranted when selecting the internal control gene for the qRT-PCR gene expression studies. Here, we provide a template of stable ECGs for investigation of radiation induced gene expression.
PMID: 22594372 [PubMed - as supplied by publisher]
Comprehensive expression profiling of microRNAs in laryngeal squamous cell carcinoma.
Head Neck. 2012 May 18;
Authors: Cao P, Zhou L, Zhang J, Zheng F, Wang H, Ma D, Tian J
Abstract
BACKGROUND: MicroRNAs (miRNAs) are noncoding RNAs involved in posttranscriptional regulation of gene expression in cancer and provide new perspectives on the development of laryngeal squamous cell carcinoma (SCC). METHODS: miRNA expression of 6 pairs of laryngeal SCC and adjacent normal tissues was screened using miRNA array. Laser capture microdissection was applied to isolate a homogeneous group of cells from laryngeal SCC samples. The results of miRNA array analysis were validated in 48 pairs of laryngeal SCC and adjacent normal tissues using quantitative RT-PCR. RESULTS: Twenty-nine differentially expressed miRNAs were detected in the 6 pairs of laryngeal SCC, of which 6 were confirmed, including upregulation of miR-21, miR-93, miR-205, and miR-708 and downregulation of miR-125b and miR-145. Their putative target genes were predicted using 3 online software programs. CONCLUSION: These differentially expressed miRNAs may play a role in tumorigenesis and progression in laryngeal SCC and offer new angles for further investigations into the function of miRNAs. © 2012 Wiley Periodicals, Inc. Head Neck, 2012.
PMID: 22605671 [PubMed - as supplied by publisher]
Workflows for microarray data processing in the Kepler environment.
BMC Bioinformatics. 2012 May 17;13(1):102
Authors: Stropp T, McPhillips T, Ludaescher B, Bieda M
Abstract
ABSTRACT: BACKGROUND: Microarray data analysis has been the subject of extensive and ongoing pipeline development due to its complexity, the availability of several options at each analysis step, and the development of new analysis demands, including integration with new data sources. Bioinformatics pipelines are usually custom built for different applications, making them typically difficult to modify, extend and repurpose. Scientific workflow systems are intended to address these issues by providing general-purpose frameworks in which to develop and execute such pipelines. The Kepler workflow environment is a well-established system under continual development that is employed in several areas of scientific research. Kepler provides a flexible graphical interface, featuring clear display of parameter values, for design and modification of workflows. It has capabilities for developing novel computational components in the R, Python, and Java programming languages, all of which are widely used for bioinformatics algorithm development, along with capabilities for invoking external applications and using web services. RESULTS: We developed a series of fully functional bioinformatics pipelines addressing common tasks in microarray processing in the Kepler workflow environment. These pipelines consist of a set of tools for GFF file processing of NimbleGen chromatin immunoprecipitation on microarray (ChIP-chip) datasets and more comprehensive workflows for Affymetrix gene expression microarray bioinformatics and basic primer design for PCR experiments, which are often used to validate microarray results. Although functional in themselves, these workflows can be easily customized, extended, or repurposed to match the needs of specific projects and are designed to be a toolkit and starting point for specific applications. These workflows illustrate a workflow programming paradigm focusing on local resources (programs and data) and therefore are close to traditional shell scripting or R/BioConductor scripting approaches to pipeline design. Finally, we suggest that microarray data processing task workflows may provide a basis for future example-based comparison of different workflow systems. CONCLUSIONS: We provide a set of tools and complete workflows for microarray data analysis in the Kepler environment, which has the advantages of offering graphical, clear display of conceptual steps and parameters and the ability to easily integrate other resources such as remote data and web services.
PMID: 22594911 [PubMed - as supplied by publisher]
Mycobacterium avium subsp. paratuberculosis survival during fermentation of soured milk products detected by culture and quantitative real time PCR methods.
Int J Food Microbiol. 2012 May 1;
Authors: Klanicova B, Slana I, Roubal P, Pavlik I, Kralik P
Abstract
Mycobacterium avium paratuberculosis (MAP), etiological agent of paratuberculosis in ruminants, is able to survive extreme conditions like very low pH (stomach), high temperature (pasteurization) or low temperature (refrigerated storage). Cheese, infant powder milk, cream and other milk and dairy products might thus be considered as possible sources of MAP for humans. The aim of this study was to investigate the survival of two MAP field isolates during fermentation of three different types of soured milk products (SMP; yogurt, acidophilus milk and kefir) under laboratory conditions. Pasteurized MAP-free milk was artificially contaminated with 10(6)MAPcells/mL and survival and absolute numbers of MAP were monitored during fermentation (4 or 16h) and after six weeks of storage at 4°C by culture and quantitative real time PCR (qPCR). Viability of MAP was determined by culture using Herrold's egg yolk medium and Middlebrook 7H10 with antibiotics, supplemented with Mycobactin J and incubated at 37°C for up to 12weeks. The absolute numbers of MAP were quantified by previously published qPCR assays targeting F57 and IS900 loci in MAP genome. We herein confirm that MAP can survive pH reduction, however, longer exposure to pH below 4 in SMP seems to be critical because it inhibits growth. Therefore, it is suggested that probiotic cultures that can decrease pH below 4 during fermentation could provide better inactivation of MAP in SMP.
PMID: 22591549 [PubMed - as supplied by publisher]
Identification of DNA Signatures Suitable for Developing into Real-Time PCR assays by Whole Genome Sequence Approaches: Using Streptococcus pyogenes as a pilot study.
J Clin Microbiol. 2012 May 16;
Authors: Hung GC, Nagamine K, Li B, Lo SC
Abstract
A stepwise computational approach using three layers of publicly available software was described to effectively identify DNA signatures for Streptococcus pyogenes. PCR testing validated 9 out of 15 signature-derived primer sets could detect as low as 5 fg of target DNA with high specificity. The selected signature-derived primer sets were successfully evaluated against all 23 clinical isolates. The approach is readily applicable for designing molecular assays for rapid detection and characterization of various pathogenic bacteria.
PMID: 22593599 [PubMed - as supplied by publisher]
The Combination of Quantitative PCR and Western Blot Detecting CP4-EPSPS Component in Roundup Ready Soy Plant Tissues and Commercial Soy-Related Foodstuffs.
J Food Sci. 2012 May 16;
Authors: Xiao X, Wu H, Zhou X, Xu S, He J, Shen W, Zhou G, Huang M
Abstract
With the widespread use of Roundup Ready soy (event 40-3-2) (RRS), the comprehensive detection of genetically modified component in foodstuffs is of significant interest, but few protein-based approaches have been found useful in processed foods. In this report, the combination of quantitative PCR (qPCR) and western blot was used to detect cp4-epsps gene and its protein product in different RRS plant tissues and commercial soy-containing foodstuffs. The foods included those of plant origin produced by different processing procedures and also some products containing both meat and plant protein concentrates. The validity of the 2 methods was confirmed first. We also showed that the CP4-EPSPS protein existed in different RRS plant tissues. In certain cases, the results from the western blot and the qPCR were not consistent. To be specific, at least 2 degraded fragments of CP4-EPSPS protein (35.5 and 24.6 kDa) were observed. For dried bean curd crust and deep-fried bean curd, a degraded protein fragment with the size of 24.6 kDa appeared, while cp4-epsps gene could not be traced by qPCR. In contrast, we found a signal of cp4-epsps DNA in 3 foodstuffs, including soy-containing ham cutlet product, meat ball, and sausage by qPCR, while CP4-EPSPS protein could not be detected by western blot in such samples. Our study therefore concluded that the combination of DNA- and protein-based methods would compensate each other, thus resulting in a more comprehensive detection from nucleic acid and protein levels. Practical Application: The combination of quantitative PCR (qPCR) and western blot was used to detect cp4-epsps gene and its protein product in different Roundup Ready soy (event 40-3-2) plant tissues and commercial soy-containing foodstuffs. The foods included those of plant origin produced by different processing procedures and also some products containing a combination of both meat and plant protein concentrates. This study indicated that the combination of DNA- and protein-based methods would supplement each other for genetically modified detection from nucleic acid and protein levels. Accordingly, qPCR and western blot could be used in CP4-EPSPS detection in a wide variety of soy-related foodstuffs.
PMID: 22591269 [PubMed - as supplied by publisher]
Systematic evaluation of the root cause of non-linearity in liquid chromatography/tandem mass spectrometry bioanalytical assays and strategy to predict and extend the linear standard curve range.
Rapid Commun Mass Spectrom. 2012 Jun 30;26(12):1465-74
Authors: Yuan L, Zhang D, Jemal M, Aubry AF
Abstract
RATIONALE: The linear range of a liquid chromatography/tandem mass spectrometry (LC/MS/MS) bioanalytical assay is typically about three orders of magnitude. A broader standard curve range is favored since it can significantly reduce the time, labor and potential errors related to sample dilution - one of the bottlenecks in sample analysis. Using quadratic regression to fit the standard curve can, to a certain degree, extend the dynamic range. However, the use of a quadratic regression is controversial, particularly in regulated bioanalysis.
METHODS: A number of compounds, with different physicochemical properties and ionization efficiencies, were evaluated to understand the cause of the non-linear behavior of the standard curve.
RESULTS: The standard curve behavior is primarily associated with the absolute analyte response but not the analyte concentration, the properties of the analyte, or the nature of the matrix when a stable-isotope-labeled internal standard (SIL-IS) is used. For all the test compounds, a non-linear curve was observed when signals exceeded a certain response, which depends on the detector used in the mass spectrometer. With typical API4000 instruments used for the experiments, this critical response level was determined to be ~1 E+6 counts per second (cps) and it was successfully used to predict the linear ranges for the test compounds. By simultaneously monitoring two selective reaction monitoring (SRM) channels of different intensity and using SIL-IS, a linear range of five orders of magnitude was achieved.
CONCLUSIONS: In this work, the root cause of the non-linear behavior of the standard curve when using a SIL-IS was investigated and identified. Based on the findings, an improved multiple SRM channels approach was proposed and successfully applied to obtain a linear dynamic range of five orders of magnitude for one test compound. This approach may work particularly well for LC/MS/MS bioanalytical assay of dried blood spot (DBS) samples, for which a direct dilution is cumbersome. Copyright © 2012 John Wiley & Sons, Ltd.
PMID: 22592990 [PubMed - in process]
Validation of reference genes for real-time quantitative PCR normalization in soybean developmental and germinating seeds.
Plant Cell Rep. 2012 May 16;
Authors: Li Q, Fan CM, Zhang XM, Fu YF
Abstract
Most of traditional reference genes chosen for real-time quantitative PCR normalization were assumed to be ubiquitously and constitutively expressed in vegetative tissues. However, seeds show distinct transcriptomes compared with the vegetative tissues. Therefore, there is a need for re-validation of reference genes in samples of seed development and germination, especially for soybean seeds. In this study, we aimed at identifying reference genes suitable for the quantification of gene expression level in soybean seeds. In order to identify the best reference genes for soybean seeds, 18 putative reference genes were tested with various methods in different seed samples. We combined the outputs of both geNorm and NormFinder to assess the expression stability of these genes. The reference genes identified as optimums for seed development were TUA5 and UKN2, whereas for seed germination they were novel reference genes Glyma05g37470 and Glyma08g28550. Furthermore, for total seed samples it was necessary to combine four genes of Glyma05g37470, Glyma08g28550, Glyma18g04130 and CYP for normalization. Key message We identified several reference genes that stably expressed in soybean seed developmental and germinating processes.
PMID: 22588479 [PubMed - as supplied by publisher]
Immunopathologic processes in sympathetic ophthalmia as signified by microRNA profiling.
Invest Ophthalmol Vis Sci. 2012 May 15;
Authors: Kaneko Y, Wu GS, Saraswathy S, Vasconcelos-Santos DV, Rao NA
Abstract
Purpose:Recent discovery of microRNAs and their negative gene regulation have provided a new understanding in the pathogenesis of inflammatory diseases. In this study, microRNA expression profiling and their likely role in sympathetic ophthalmia (SO) were investigated, using formalin-fixed, paraffin-embedded samples. Methods:Two groups of four enucleated globes (total eight globes) from patients with clinical and histopathological diagnosis of SO (experimental samples) and 1 group of four age-matched, non-inflamed enucleated globes (control samples) were used. Human genome-wide microRNA polymerase chain reaction (PCR) array was performed and results were subjected to bioinformatics calculation and P-values stringency tests. The targets were searched using the recently published and periodically updated miRWalk software. Quantitative real-time PCR and immunohistochemical staining were performed to confirm the validated targets in the mRNA and in the protein levels respectively.Results:No microRNA was significantly upregulated, but 27 microRNAs were significantly downregulated. Among these, four microRNAs (hsa-miR-1, hsa-let-7e, hsa-miR-9, and hsa-miR-182) were known to be associated with inflammatory signaling pathway. Only hsa-miR-9 has the validated targets, tumor necrosis factor-α and nuclear factor kappa B1, which have been previously shown to be associated with mitochondrial oxidative stress-mediated photoreceptor apoptosis in eyes with SO.Conclusions:Identification of altered levels of microRNAs by microRNA expression profiling may yield new insights into the pathogenesis of SO by disclosing specific microRNAs signatures. In the future these may be targeted by synthetic microRNA mimic-based therapeutic strategies.
PMID: 22589448 [PubMed - as supplied by publisher]
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