In Situ Hybridization for mRNA Localization Purpose: In Situ hybridization for mRNA localization is used to identify the position of target mRNA in the cell or tissues being examined(Smith, 2001).
Methods: In order for in situ hybridization for localization of mRNA to be successful, specific modifications to the generic protocol must be made in each circumstance in order to ensure accurate labeling of the desired structures for observation. The cells which contain the mRNA and associated structures of interest are cultured and fixed to a poly-L-lysine coated microscopic slide(Raap et al, 1991). The cells are then permeabilized so that the probe will be able to penetrate the tissues and attach to the target sequence (Raap et al, 1991). This is achieved by a series of ethanol washes, treatment in xylene to remove residual lipids, incubation in PBS, treatment in pepsin in N HCl at 37 C and treatment in formaldehyde for 10 minutes followed by another wash with PBS(Raap et al, 1991).
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Ethel Smith (left) and Fanny Rosenfeld (second fro...Prehybridization, carried out to reduce background staining, is composed of the hybridization solution, minus the probe. The probe, a complementary strand of nucleotide, is labeled by nick translation and a reporter molecule is attached (Raap et al, 1991). The reporter molecule could be digoxigenen, biotin or fluorochromes (Raap et al, 1991). It is then denatured at 80 C and mixed with the hybridization solution (Raap et al, 1991). This solution contains deionized formamide to reduce thermal stability of bonds to allow a lower hybridization temperature, NaCl and sodium citrate to decrease electrostatic interactions between the two strands, EDTA to remove free divalent cations from the solution, NaH PO buffer, dextran sulphate and sheared salmon sperm DNA to decrease nonspecific attachments of the probe(Smith, 2001). This mixture is poured on the slide and it is left for 16 hours...