This protocol has several roots. It is based on a protocol to create cDNA for microarrays and adapted to be used for lower amounts. It works very reliable and it is my preferred way of cDNA synthesis.
To study objects we must interact with them. Generally, the technique of interaction is determined by the size of the object. Thus, an object of macroscopic size can be studied by direct contact. However, microscopic objects, such as the cell and its organelles, must be studied with agents of similar or smaller size. Cells in are roughly 3–30 microns in diameter, thus, light with wavelength in the visible range (~500 nm) is an ideal interacting agent. The set up of light microscopes affords a resolution that is about half the wavelength of light employed. Thus, light microscopy is useful for examining cells and cellular substructures on the order of 200–300 nm or larger. However, vesicles (often 50 nm in diameter), and other objects of similar or smaller scale cannot be resolved using current setups. Although, in principle, light of very short wavelength (e.g. X-rays) could be used to resolve smaller cellular structures, such light is too energetic, damaging the cell upon contact. In addition, lenses to focus high energy photons do not exist. High resolution can be obtained using electron microscopy. Moving electrons in an electron microscope possess wavelengths on the order of 0.3 nm. Using an electron microscope, the electrons can be used to form resolved images of cellular structures of about 3 nm in size.
Vortex the lysate for 1 min and pass through a QIAshredder column (Ambion). The lysate can be stored at 80°C overnight. RNA quality decreases, however, with the extended storage. Isolate total RNA with the RNeasy Mini kit with the DNase I set (Ambion).
Most somatic cell deaths occur during embryogenesis and in wild-type animals the cell corpses are engulfed and rapidly degraded. Therefore, cell corpses are rarely found in the head region of freshly hatched L1 larvae (; ; ). In animals, however, engulfment is blocked, resulting in the accumulation of cell corpses in L1 larvae (about 23 corpses). If cell death is blocked in this mutant background, for example by a (lf) mutation, few or no cell corpses are detected (; (Ellis et al., 1991). Therefore, this assay can also be used to determine if cells that are destined to undergo programmed cell death failed to die.
SYTO dyes (Molecular Probes) clearly label DNA in all cells. However, condensed DNA in apoptotic cells (“dead nuclei”) stains more brightly than DNA in living cells. SYTO11, SYTO12, and Acridine Orange have given the best results (; ; ).
TUNEL (dT-mediated dTP ick nd abeling) labels DNA ends, and therefore also dying cells, in which DNA becomes degraded (). TUNEL can be used as a marker for dying cells and to analyze DNA degradation. DNA degradation occurs very rapidly during cell death, thus, TUNEL stains apoptotic cells only during a transient stage of DNA degradation. For this reason, usually only a small subset of corpses is labeled with this technique. However, in mutants, which are defective in some aspects of DNA degradation, the nuclei of many dying cells can be labeled using this method ().
This ready-to-use dNTP Mix is a mixture of all four nucleotides (dATP, dCTP, dGTP, dTTP), each dissolved in highly purified water at a concentration of 2.5 mM, at pH 8.3. Two milliliters of the Mix is enough for 500 standard 50 µl and DNA Polymerase PCR reactions.
Cellular structures and components can be labeled with a variety of fluorescent dyes that can be visualized using a fluorescence microscope. In response to incident light of specified wavelength (produced by a laser or using appropriate filters), fluorescent dye molecules are excited. Decay of the molecule from the excited state to an intermediate excited state produces a photon of a wavelength longer than that of the excitation source, which can easily be detected and separated, using appropriate filters, from the incident light (for a more detailed description see ). Below are a number of protocols for using dyes to visualize DNA, proteins and cellular structures. Dyes coupled to antibodies are particularly useful for visualizing the presence and localization of specific proteins within the cell, and several methods for antibody staining are discussed in the chapter on gene expression. Often it is of interest to study whether two cellular molecules physically interact. Fluorescence resonance energy transfer (FRET) has been used to indicate the proximity of two labeled fluorophores. However, this method has not been significantly used in to date.
The performance of ProtoScript First Strand cDNA Synthesis Kit is tested in an RT reaction using human Jurkat total RNA with primer d(T)23VN. The sensitivity of the kit is verified by the detection of GAPDH transcript in 20 pg total RNA after 35 cycles. The length of cDNA achieved is verified by the detection of a 5.5 kb amplicon of the p532 gene.
This high-purity dNTP set allows you to formulate your own PCR mix. Each of all four nucleotides (dATP, dCTP, dGTP, dTTP), is provided in a separate vial (750 µl each), dissolved at a concentration of 10 mM in highly purified water, pH 8.3.