Identification of suitable internal control genes for expression studies in Coffea arabica under different experimental conditions

<p>Abstract</p> <p>Background</p> <p>Quantitative data from gene expression experiments are often normalized by transcription levels of reference or housekeeping genes. An inherent assumption for their use is that the expression of these genes is highly uniform in living organisms during various phases of development, in different cell types and under diverse environmental conditions. To date, the validation of reference genes in plants has received very little attention and suitable reference genes have not been defined for a great number of crop species including <it>Coffea arabica</it>. The aim of the research reported herein was to compare the relative expression of a set of potential reference genes across different types of tissue/organ samples of coffee. We also validated the expression profiles of the selected reference genes at various stages of development and under a specific biotic stress.</p> <p>Results</p> <p>The expression levels of five frequently used housekeeping genes (reference genes), namely <it>alcohol dehydrogenase </it>(<it>adh</it>), <it>14-3-3</it>, <it>polyubiquitin </it>(<it>poly</it>), <it>β-actin </it>(<it>actin</it>) and <it>glyceraldehyde-3-phosphate dehydrogenase </it>(<it>gapdh</it>) was assessed by quantitative real-time RT-PCR over a set of five tissue/organ samples (root, stem, leaf, flower, and fruits) of <it>Coffea arabica </it>plants. In addition to these commonly used internal controls, three other genes encoding a cysteine proteinase (<it>cys</it>), a caffeine synthase (<it>ccs</it>) and the 60S ribosomal protein L7 (<it>rpl7</it>) were also tested. Their stability and suitability as reference genes were validated by geNorm, NormFinder and BestKeeper programs. The obtained results revealed significantly variable expression levels of all reference genes analyzed, with the exception of <it>gapdh</it>, which showed no significant changes in expression among the investigated experimental conditions.</p> <p>Conclusion</p> <p>Our data suggests that the expression of housekeeping genes is not completely stable in coffee. Based on our results, <it>gapdh</it>, followed by <it>14-3-3 </it>and <it>rpl7 </it>were found to be homogeneously expressed and are therefore adequate for normalization purposes, showing equivalent transcript levels in different tissue/organ samples. <it>Gapdh </it>is therefore the recommended reference gene for measuring gene expression in <it>Coffea arabica</it>. Its use will enable more accurate and reliable normalization of tissue/organ-specific gene expression studies in this important cherry crop plant.</p

Evidence of a noncoding transcript of the RIPK2 gene overexpressed in head and neck tumor

Receptor-interacting proteins are a family of serine/threonine kinases, which integrate extra and intracellular stress signals caused by different factors, including infections, inflammation and DNA damage. Receptor-interacting serine/threonine-protein kinase 2 (RIP-2) is a member of this family and an important component of the nuclear factor NF-kappa-B signaling pathway. The corresponding human gene RIPK2 generates two transcripts by alternative splicing, the full-length and a short transcript. The short transcript has a truncated 5’ sequence, which results in a predicted isoform with a partial kinase domain but able to transduce signals through its caspase recruitment domain. In this study, the expression of RIPK2 was investigated in human tissue samples and, in order to determine if both transcripts are similarly regulated at the transcriptional level, cancer cell lines were submitted to temperature and acid stresses. We observed that both transcripts are expressed in all tissues analyzed, with higher expression of the short one in tumor samples, and they are differentially regulated following temperature stress. Despite transcription, no corresponding protein for the short transcript was detected in tissues and cell lines analyzed. We propose that the shorter transcript is a noncoding RNA and that its presence in the cell may play regulatory roles and affect inflammation and other biological processes related to the kinase activity of RIP-2.Instituto de Biotecnologia y Biologia Molecula

A Comparative Study of Head-Mounted and Table-Mounted Augmented Vision Systems for Assembly Error Detection

It is proposed a simple and inexpensive strategy to determine singlet oxygen (¹O2) quantum yields (&#934;&#916;) of photosensitizers (PS) in water using beetroot extract containing betacyanin (Bc) and a set of light emitting diodes (LEDs) for excitation. Bc, a cationic natural dye, was obtained by flash chromatography purification from the red beet extract (Beta vulgaris) and employed as a convenient probe for ¹O2 detection. Solutions of Bc and PS were illuminated with an array of LEDs adapted in the cuvette compartment of a commercial spectrophotometer, and the decrease in Bc absorbance was followed as a function of time. Bc photobleaching decreased in de-aerated solution and increased in D2O, indicating the involvement of ¹O2. The observed photobleaching rate constant (k obs) was proportional to the LED intensity, concentration and &#934;&#916; of the PS. By keeping the light source constant we could estimate the overlap integral (R) between the LED emission and PS absorbance for different PS concentrations. The slope of R versus k obs is the value of the photobleaching rate constant (k), which was shown to be proportional to &#934;&#916;. Values of &#934;&#916; obtained by this method were compared with those obtained by measuring NIR (near infrared) emission for a series phenothiazine dyes.É proposta uma estratégia simples e barata para determinar rendimentos quânticos (&#934;&#916;) de oxigênio singlete (¹O2) de fotossensibilizadores (PS) em água utilizando extrato de beterraba contendo betacianina (Bc) e um conjunto de diodos emissores de luz (LEDs) para excitação. Bc, um corante catiônico natural, foi obtida por purificação através de cromatografia a partir do extrato de beterraba vermelha (Beta vulgaris), e utilizada como uma sonda para detecção de ¹O2. Soluções do Bc e PS foram iluminadas com um arranjo de LEDs adaptado no compartimento de um espectrofotômetro comercial e a diminuição da absorvância de Bc foi seguida em função do tempo. O fotobranqueamento de Bc diminuiu em solução purgada com nitrogênio e aumentou em D2O, indicando o envolvimento de ¹O2. A constante de velocidade de fotobranqueamento observada (k obs) foi proporcional à intensidade do LED, à concentração e ao &#934;&#916; do PS. Mantendo a fonte de luz constante pudemos estimar a integral de sobreposição (R) entre a absorção do PS e a emissão do LED para diferentes concentrações de PS. A inclinação da curva de R em função de k obs é o valor da constante de velocidade de fotobranqueamento (k), que foi mostrada ser proporcional a &#934;&#916;. Valores de &#934;&#916; obtidos por este método foram comparados com aqueles obtidos através da medição da emissão no NIR (infravermelho próximo) para uma série de corantes fenotiazínicos.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Rede de Nanotecnologia Molecular e InterfacesInstituto do Milênio de Materiais Complexo

Partial molecular characterization of the Nile tilapia (Oreochromis niloticus) alpha-cardiac muscle actin gene and its relationship to actin isoforms of other fish species

An alpha actin gene segment, isolated from Nile tilapia (Oreochromis niloticus), was characterized by nucleotide sequencing, predicted amino acid sequence and Southern blot hybridization. Genomic DNA amplification resulted in a 1063-bp fragment corresponding to a partial alpha-cardiac muscle actin gene containing exons 3 to 6. Southern blot analysis of the restriction-digested DNA revealed that the Nile tilapia genome contains multiple muscle actin isoforms. Although comparison of the nucleotide sequence, amino acid residues and exon-intron organization of the isolated actin gene with those of other vertebrates showed a high level of identity, diagnostic amino acid residues can still be correlated to distinct actin genes in fish species