Transcriptomic Analysis of Starch Biosynthesis in the Developing Grain of Hexaploid Wheat

The expression of genes involved in starch synthesis in wheat was analyzed together with the accumulation profiles of soluble sugars, starch, protein, and starch granule distribution in developing caryopses obtained from the same biological materials used for profiling of gene expression using DNA microarrays. Multiple expression patterns were detected for the different starch biosynthetic gene isoforms, suggesting their relative importance through caryopsis development. Members of the ADP-glucose pyrophosphorylase, starch synthase, starch branching enzyme, and sucrose synthase gene families showed different expression profiles; expression of some members of these gene families coincided with a period of high accumulation of starch while others did not. A biphasic pattern was observed in the rates of starch and protein accumulation which paralleled changes in global gene expression. Metabolic and regulatory genes that show a pattern of expression similar to starch accumulation and granule size distribution were identified, suggesting their coinvolvement in these biological processes

Selection of reference genes for gene expression studies in ultraviolet B-irradiated human skin fibroblasts using quantitative real-time PCR

<p>Abstract</p> <p>Background</p> <p>Reference genes are frequently used to normalise mRNA levels between different samples. The expression level of these genes, however, may vary between tissues or cells and may change under certain circumstances. Cytoskeleton genes have served as multifunctional tools for experimental studies as reference genes. Our previous studies have demonstrated that the expression of vimentin, one cytoskeletal protein, was increased in ultraviolet B (UVB)-irradiated fibroblasts. Thus, we examined the expression of other cytoskeleton protein genes, <it>ACTB </it>(<it>actin, beta</it>), <it>TUBA1A </it>(<it>tubulin, alpha 1a</it>), and <it>TUBB1 </it>(<it>tubulin, beta 1</it>), in human dermal fibroblasts irradiated by UVB to determine which of these candidates were the most appropriate reference genes.</p> <p>Results</p> <p>Quantitative real-time PCR followed by analysis with the NormFinder and geNorm software programmes was performed. The initial screening of the expression patterns demonstrated that the expression of <it>VIM </it>was suppressed after UVB irradiation at doses ≥25 mJ/cm²and that the expression of <it>TUBA1A </it>was significantly reduced by UVB doses ≥75 mJ/cm²in cultured human dermal fibroblasts. The analysis of the experimental data revealed <it>ACTB </it>to be the most stably expressed gene, followed by <it>GAPDH </it>(<it>aglyceraldehyde-3-phosphate dehydrogenase</it>), under these experimental conditions. By contrast, <it>VIM </it>was found to be the least stable gene. The combination of <it>ACTB </it>and <it>TUBB1 </it>was revealed to be the gene pair that introduced the least systematic error into the data normalisation.</p> <p>Conclusion</p> <p>The data herein provide evidence that <it>ACTB </it>and <it>TUBB1 </it>are suitable reference genes in human skin fibroblasts irradiated by UVB, whereas <it>VIM </it>and <it>TUBA1A </it>are not and should therefore be excluded as reference genes in any gene expression studies involving UVB-irradiated human skin fibroblasts.</p

Identification of Phosphoglycerate Kinase 1 (PGK1) as a reference gene for quantitative gene expression measurements in human blood RNA

<p>Abstract</p> <p>Background</p> <p>Blood is a convenient sample and increasingly used for quantitative gene expression measurements with a variety of diseases including chronic fatigue syndrome (CFS). Quantitative gene expression measurements require normalization of target genes to reference genes that are stable and independent from variables being tested in the experiment. Because there are no genes that are useful for all situations, reference gene selection is an essential step to any quantitative reverse transcription-PCR protocol. Many publications have described appropriate genes for a wide variety of tissues and experimental conditions, however, reference genes that may be suitable for the analysis of CFS, or human blood RNA derived from whole blood as well as isolated peripheral blood mononuclear cells (PBMCs), have not been described.</p> <p>Findings</p> <p>Literature review and analyses of our unpublished microarray data were used to narrow down the pool of candidate reference genes to six. We assayed whole blood RNA from Tempus tubes and cell preparation tube (CPT)-collected PBMC RNA from 46 subjects, and used the geNorm and NormFinder algorithms to select the most stable reference genes. <it>Phosphoglycerate kinase 1 (PGK1) </it>was one of the optimal normalization genes for both whole blood and PBMC RNA, however, additional genes differed for the two sample types; <it>Ribosomal protein large, P0 (RPLP0</it>) for PBMC RNA and <it>Peptidylprolyl isomerase B </it>(<it>PPIB) </it>for whole blood RNA. We also show that the use of a single reference gene is sufficient for normalization when the most stable candidates are used.</p> <p>Conclusions</p> <p>We have identified <it>PGK1 </it>as a stable reference gene for use with whole blood RNA and RNA derived from PBMC. When stable genes are selected it is possible to use a single gene for normalization rather than two or three. Optimal normalization will improve the ability of results from PBMC RNA to be compared with those from whole blood RNA and potentially allows comparison of gene expression results from blood RNA collected and processed by different methods with the intention of biomarker discovery. Results of this study should facilitate large-scale molecular epidemiologic studies using blood RNA as the target of quantitative gene expression measurements.</p

RefGenes: identification of reliable and condition specific reference genes for RT-qPCR data normalization

Background RT-qPCR is a sensitive and increasingly used method for gene expression quantification. To normalize RT-qPCR measurements between samples, most laboratories use endogenous reference genes as internal controls. There is increasing evidence, however, that the expression of commonly used reference genes can vary significantly in certain contexts. Results Using the Genevestigator database of normalized and well-annotated microarray experiments, we describe the expression stability characteristics of the transciptomes of several organisms. The results show that a) no genes are universally stable, b) most commonly used reference genes yield very high transcript abundances as compared to the entire transcriptome, and c) for each biological context a subset of stable genes exists that has smaller variance than commonly used reference genes or genes that were selected for their stability across all conditions. Conclusion We therefore propose the normalization of RT-qPCR data using reference genes that are specifically chosen for the conditions under study. RefGenes is a community tool developed for that purpose. Validation RT-qPCR experiments across several organisms showed that the candidates proposed by RefGenes generally outperformed commonly used reference genes. RefGenes is available within Genevestigator at http://www.genevestigator.com

The effects of hypertonic fluid administration on the gene expression of inflammatory mediators in circulating leucocytes in patients with septic shock: a preliminary study

Contains fulltext : 98426.pdf (publisher's version ) (Open Access)ABSTRACT: OBJECTIVE: This study was designed to investigate the effect of hypertonic fluid administration on inflammatory mediator gene expression in patients with septic shock. DESIGN AND SETTING: Prospective, randomized, controlled, double-blind clinical study in a 15-bed mixed intensive care unit in a tertiary referral teaching hospital. INTERVENTIONS: Twenty-four patients, who met standard criteria for septic shock, were randomized to receive a bolus of hypertonic fluid (HT, 250 ml 6% HES/7.2% NaCl) or isotonic fluid (IT, 500 ml 6% HES/0.9% NaCl) administered over 15 minutes. Randomization and study fluid administration was within 24 hours of ICU admission for all patients. This trial is registered with ANZCTR.org.au as ACTRN12607000259448. RESULTS: Blood samples were taken immediately before and 4, 8, 12, and 24 hours after fluid administration. Real-time reverse transcriptase polymerase chain reaction (RT rtPCR) was used to quantify mRNA expression of different inflammatory mediators in peripheral leukocytes. In the HT group, compared with the IT group, levels of gene expression of MMP9 and L-selectin were significantly suppressed (p = 0.0002 and p = 0.007, respectively), and CD11b gene expression tended to be elevated (p = NS). No differences were found in the other mediators examined. CONCLUSIONS: In septic shock patients, hypertonic fluid administration compared with isotonic fluid may modulate expression of genes that are implicated in leukocyte-endothelial interaction and capillary leakage.The study was performed at the Intensive Care Department, Waikato Hospital, and at the Molecular Genetics Laboratory, University of Waikato, Hamilton, New Zealand. TRIAL REGISTRATION: Australia and New Zealand Clinical Trials Register (ANZCTR): ACTRN12607000259448

Identification and validation of suitable endogenous reference genes for gene expression studies in human peripheral blood

Background Gene expression studies require appropriate normalization methods. One such method uses stably expressed reference genes. Since suitable reference genes appear to be unique for each tissue, we have identified an optimal set of the most stably expressed genes in human blood that can be used for normalization. Methods Whole-genome Affymetrix Human 2.0 Plus arrays were examined from 526 samples of males and females ages 2 to 78, including control subjects and patients with Tourette syndrome, stroke, migraine, muscular dystrophy, and autism. The top 100 most stably expressed genes with a broad range of expression levels were identified. To validate the best candidate genes, we performed quantitative RT-PCR on a subset of 10 genes (TRAP1, DECR1, FPGS, FARP1, MAPRE2, PEX16, GINS2, CRY2, CSNK1G2 and A4GALT), 4 commonly employed reference genes (GAPDH, ACTB, B2M and HMBS) and PPIB, previously reported to be stably expressed in blood. Expression stability and ranking analysis were performed using GeNorm and NormFinder algorithms. Results Reference genes were ranked based on their expression stability and the minimum number of genes needed for nomalization as calculated using GeNorm showed that the fewest, most stably expressed genes needed for acurate normalization in RNA expression studies of human whole blood is a combination of TRAP1, FPGS, DECR1 and PPIB. We confirmed the ranking of the best candidate control genes by using an alternative algorithm (NormFinder). Conclusion The reference genes identified in this study are stably expressed in whole blood of humans of both genders with multiple disease conditions and ages 2 to 78. Importantly, they also have different functions within cells and thus should be expressed independently of each other. These genes should be useful as normalization genes for microarray and RT-PCR whole blood studies of human physiology, metabolism and disease.Boryana S Stamova, Michelle Apperson, Wynn L Walker, Yingfang Tian, Huichun Xu, Peter Adamczy, Xinhua Zhan, Da-Zhi Liu, Bradley P Ander, Isaac H Liao, Jeffrey P Gregg, Renee J Turner, Glen Jickling, Lisa Lit and Frank R Shar

A Novel Modular Antigen Delivery System for Immuno Targeting of Human 6-sulfo LacNAc-Positive Blood Dendritic Cells (SlanDCs)

Previously, we identified a major myeloid-derived proinflammatory subpopulation of human blood dendritic cells which we termed slanDCs (e.g. Schäkel et al. (2006) Immunity 24, 767-777). The slan epitope is an O-linked sugar modification (6-sulfo LacNAc, slan) of P-selectin glycoprotein ligand-1 (PSGL-1). As slanDCs can induce neoantigen-specific CD4+ T cells and tumor-reactive CD8+ cytotoxic T cells, they appear as promising targets for an in vivo delivery of antigens for vaccination. However, tools for delivery of antigens to slanDCs were not available until now. Moreover, it is unknown whether or not antigens delivered via the slan epitope can be taken up, properly processed and presented by slanDCs to T cells.Single chain fragment variables were prepared from presently available decavalent monoclonal anti-slan IgM antibodies but failed to bind to slanDCs. Therefore, a novel multivalent anti-slanDC scaffold was developed which consists of two components: (i) a single chain bispecific recombinant diabody (scBsDb) that is directed on the one hand to the slan epitope and on the other hand to a novel peptide epitope tag, and (ii) modular (antigen-containing) linker peptides that are flanked at both their termini with at least one peptide epitope tag. Delivery of a Tetanus Toxin-derived antigen to slanDCs via such a scBsDb/antigen scaffold allowed us to recall autologous Tetanus-specific memory T cells.In summary our data show that (i) the slan epitope can be used for delivery of antigens to this class of human-specific DCs, and (ii) antigens bound to the slan epitope can be taken up by slanDCs, processed and presented to T cells. Consequently, our novel modular scaffold system may be useful for the development of human vaccines

Correlations of Gene Expression with Blood Lead Levels in Children with Autism Compared to Typically Developing Controls

The objective of this study was to examine the correlation between gene expression and lead (Pb) levels in blood in children with autism (AU, n = 37) compared to typically developing controls (TD, n = 15). We postulated that, though lead levels did not differ between the groups, AU children might metabolize lead differently compared to TD children. RNA was isolated from blood and processed on Affymetrix microarrays. Separate analyses of covariance (ANCOVA) corrected for age and gender were performed for TD, AU, and all subjects (AU + TD). To reduce false positives, only genes that overlapped these three ANCOVAs were considered. Thus, 48 probe sets correlated with lead levels in both AU and TD subjects and were significantly different between the groups (p(Diagnosis × log2 Pb) < 0.05). These genes were related mainly to immune and inflammatory processes, including MHC Class II family members and CD74. A large number (n = 791) of probe sets correlated (P ≤ 0.05) with lead levels in TD but not in AU subjects; and many probe sets (n = 162) correlated (P ≤ 0.05) with lead levels in AU but not in TD subjects. Only 30 probe sets correlated (P ≤ 0.05) with lead levels in a similar manner in the AU and TD groups. These data show that AU and TD children display different associations between transcript levels and low levels of lead. We postulate that this may relate to the underlying genetic differences between the two groups, though other explanations cannot be excluded

Correlations Between Gene Expression and Mercury Levels in Blood of Boys With and Without Autism

Gene expression in blood was correlated with mercury levels in blood of 2- to 5-year-old boys with autism (AU) compared to age-matched typically developing (TD) control boys. This was done to address the possibility that the two groups might metabolize toxicants, such as mercury, differently. RNA was isolated from blood and gene expression assessed on whole genome Affymetrix Human U133 expression microarrays. Mercury levels were measured using an inductively coupled plasma mass spectrometer. Analysis of covariance (ANCOVA) was performed and partial correlations between gene expression and mercury levels were calculated, after correcting for age and batch effects. To reduce false positives, only genes shared by the ANCOVA models were analyzed. Of the 26 genes that correlated with mercury levels in both AU and TD boys, 11 were significantly different between the groups (P(Diagnosis*Mercury) ≤ 0.05). The expression of a large number of genes (n = 316) correlated with mercury levels in TD but not in AU boys (P ≤ 0.05), the most represented biological functions being cell death and cell morphology. Expression of 189 genes correlated with mercury levels in AU but not in TD boys (P ≤ 0.05), the most represented biological functions being cell morphology, amino acid metabolism, and antigen presentation. These data and those in our companion study on correlation of gene expression and lead levels show that AU and TD children display different correlations between transcript levels and low levels of mercury and lead. These findings might suggest different genetic transcriptional programs associated with mercury in AU compared to TD children