InSitu Hybridization For MRNA Localization

In Situ Hybridization for mRNA Localization Purpose: In Situ hybridisation for mRNA localization is used to identify the position of organize 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 do in each circumstance in order to keep in line accurate labeling of the desired structures for observation. The cells which contain the mRNA and associated structures of interest atomic number 18 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 qualified to penetrate the tissues and attach to the target sequence (Raap et al, 1991). This is achieved by a series of ethanol swooshes, treatment in xylene to sequestrate 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). Prehybridization, carried out to reduce background staining, is composed of the hybridization firmness of purpose, minus the probe. The probe, a complementary strand of nucleotide, is labeled by mountain pass translation and a newsperson molecule is attached (Raap et al, 1991). The reporter molecule could be digoxigenen, biotin or fluorochromes (Raap et al, 1991). It is then change 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 mitigate 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 at 37 C to allow hybridization to occur(Raap et al, 1991). A blocking solution, to remove of non specific binding, consisting of TRIS-HCl...

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