You are here: Research group of Comparative Endocrinology Research Techniques

Techniques

 

This is a summary of the most frequently used research techniques in our lab.

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Morpholino antisense technology. Morpholinos are synthetic molecules, usually 25 bases in length, which bind to complementary sequences of RNA. Morpholinos are used as a tool for reverse genetics by knocking down gene function. Morpholinos block the progression of the translation initiation complex and hence inhibit the translation of the coding region of the mRNA. Morpholino oligos are used to investigate the role of a specific mRNA transcript in the development of an embryo. We inject morpholino oligos into the yolk of 2-4 cell zebrafish embryos, producing morphant embryos to investigate the role of different components of the thyroidal axis in embryonic development.

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In vivo electroporation. Electroporation or electropermeabilization is a significant increase in the electrical conductivity and permeability of the cell plasma membrane caused by an externally applied electrical field. It is usually used in molecular biology as a way of introducing some substance into a cell, such as loading it with a molecular probe, a drug that can change the cell's function, or a piece of coding DNA. We use it in vivo to introduce vectors carrying RNAi constructs or coding sequences to silence or respectively overexpress thyroid hormone transporters or deiodinases in specific regions of the developing chicken brain. To increase the accessibility to the embryonic brain, embryos can be cultured ex ovo from day 2.5 of development onwards.

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Cell culture. Cell culture is the term applied when growing cells in an in vitro environment. Strictly speaking, this can apply to either prokaryotic or eukaryotic cells, though in practice cell culture has come to refer to the culturing of cells derived from multicellular eukaryotes, especially animal cells. Cells taken directly from animal tissues normally have only a limited life span in culture (primary cell culture) but a variety of established cell lines is available which can be cultured for a much longer time if they are split regularly in fresh growth medium. We use primary cultures of embryonic brain cells to study the endocrine factors regulating their growth. Avian and mammalian cell lines are used mainly to study regulation of gene expression via promoter-reporter constructs.

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Immunohistochemistry. The localization of proteins in a tissue can be determined by an immunohistochemical staining of tissue sections. This localization method is based on the specific recognition of the protein (antigen) by a primary (poly- or monoclonal) antibody. Cells in which the primary antibody recognizes and binds its antigen can be visualized subsequently in different ways. For example, the primary antibody can be recognized by a secondary antibody coupled to biotin; this complex will react with streptavidin-peroxidase and form a brown precipitate after the addition of the chromogen DAB. Alternatively the secondary antibody is directly or indirectly coupled to a fluorophore and the tissue section can be analysed by confocal microscopy

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In situ hybridization. In situ hybridization is used to localize the expression sites of certain mRNAs (or other nucleic acids) in a tissue. This method is based on the hybridization of the specific mRNA in the cells of the tissue with an added labelled probe. We use either radioactively labelled oligonucleotides or riboprobes, that can be detected with a photographic emulsion (dipping), or non-radioactive digoxigenin (DIG)-labelled riboprobes, that are detected by immunohistochemistry. In situ hybridization can be combined with an immunohistochemical staining of a desired cell type.

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PCR and cloning. PCR (polymerase chain reaction) is a technique that allows us to amplify a given DNA fragment exponentially, using short oligonucleotide primers that recognize certain regions in the target DNA. The PCR protocol can be preceded by a reverse transcriptase reaction in which RNA, isolated from tissues or cells, is reverse transcribed into cDNA. RT-PCR can be used to detect the expression of (even rare) mRNAs in different tissues or to clone a certain DNA sequence. The amplified DNA fragment is excised from the electrophoresis gel and transformed into bacterial cells for further amplification and sequencing.

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Real-time quantitative PCR. Real-time PCR is used to quantify gene expression in a very sensitive, reproducible and specific way. It has several advantages over other RT-PCR methods that use the amount of final amplified product (end-point) to quantify. Data are collected throughout the PCR process as it occurs, hereby combining amplification and detection in one single step. A variety of different fluorescent chemistries are available today from binding dyes to highly specific probes. During the process of real-time PCR the fluorescence emitted during the reaction is monitored and is correlated to the amount of PCR product formed. In our lab, real-time PCR is used for relative quantification of gene expression.

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Blotting techniques. In Northern blotting RNA is separated by electrophoresis and then transferred to a membrane. This membrane is then incubated with a labelled DNA- or RNA-probe, complementary to the mRNA that we wish to detect. The pattern of bands that is obtained by this method is scanned and analyzed using computer software. Northern analyse allows us to detect mRNA expression, measure it semi-quantitatively, and search for possible splice variants. Western blotting is a similar technique for proteins. After electrophoresis and transfer to a membrane, the desired proteins are visualized using an immunological staining with a specfic antibody. Western blotting is used for the detection and (semi-)quantification of proteins.

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2D-DIGE (collaboration with the lab of Neuroplasticity and Neuroproteomics). In two-dimensional differential gel electrophoresis (2D-DIGE) two protein samples are labelled with a different fluorescent dye. The samples are then mixed together and separated on the same two-dimensional gel. The first separation is based on the iso-electric point of the proteins (pH); the second is based on the molecular weight. Because both dyes emit light of a different golf length after excitation, the two protein samples can be detected independently in the same gel. Both pictures can be combined digitally and colour differences will indicate which proteins are differentially expressed.

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Kinetic in vitro enzyme tests. In our lab in vitro enzymatic tests were developed to quantify the activity of each of the three types of deiodinases. In each test protocol the preferred substrate of the concerning deiodinase is used, together with chemicals that inhibit the activity of the other deiodinase types. The reaction products are separated by high performance liquid chromatography (HPLC). Using a sulfation test, we measure the in vitro activity of sulfotransferase enzymes, present in de cytosol of the cells. Based on ion exchange chromatography, the end products, sulfated and non-sulfated substrate, are separated.

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Perifusion and static in vitro incubation. Tissues to be investigated are placed in perifusion chambers, through which a constant current of culture medium is maintained. By adding a hormone to the medium, we can study the dynamics (amplitude and duration) of the in vitro response of the tissue to the hormone. We can also incubate the tissue statically in the medium in multiwell plates. The longer contact between the tissue and the culture medium containing the hormone, will concentrate the tissue's secrete. This method, however, gives no information about the dynamics of the response as obtained with the perifusion set-up.

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Radio immunoassay (RIA). RIAs are used to quantify hormone levels in plasma or tissue extracts. The tissues are homogenized and the hormone is extracted using a suitable solute. For RIA quantification a specific antibody against the hormone to be measured is necessary, and so is the radiolabelled hormone (tracer). During an incubation step the radiolabelled hormone will compete with the endogenous hormone in the sample to bind the specific antibody. The more endogenous hormone present in the sample, the more radiolabelled hormone will be displaced from the antibody.

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Enzyme-linked immunosorbent assay (ELISA). The ELISA technique is somewhat similar to the RIA. There's also a competition for binding a specific antibody between the endogenous antigen in the sample and a fixed amount of added antigen. To make the separation of the free and antibody-bound antigen easier, a microtiter plate is used as a fixed carrier. The detection method in the ELISA, however, is not based on radioactivity, but on an enzymatic colorigenic reaction. Usually the enzyme is attached to a secondary antibody that recognizes the primary antibody.

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Radioreceptor assay (RRA). In a RRA the amount and affinity of a certain receptor type in a tissue is measured. Firstly subcellular fractions (like cell membrane fractions or nuclear fractions) are prepared. A known ammount of such a fraction is incubated with a fixed amount of radioactively labelled hormone and an increasing concentration series of unlabelled hormone. Subsequently, free and receptor-bound hormone are separated. From the resulting displacement curve the amount of receptor (Bmax) and its affinity for the hormone (Ka) can be calculated.