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While potential levels of regulation are infinite, they are RNA Methodologies Probe Target Radiolabeled antisense RNA Total cellular RNA or poly(A)+ RNA High stringency solution hybridization Hybrid formation X-ray film RNase digestion of all nonhybridized nucleic acids Gel Protected fragment Polyacrylamide Gel Electrophoresis (PAGE) 1 2 3 4 Autoradiography directly from the gel Fig. 4 RNase protection assay for the quantification of specific RNA species. Purified RNA is hybridized in solution with a labeled antisense probe sequence to form thermodynamically stable double-stranded RNA molecules.

The method of cell lysis will determine the extent of subcellular disruption in a sample, and is a direct function of the lysis buffer. For example, a lysis buffer that is used successfully with tissue culture cells may be entirely inappropriate for whole-tissue samples due to the presence of a cell wall (in the case of plants and yeast) or tenacious proteins found in the extracellular matrix (in animal tissues). The method by which membrane solubilization is accomplished will also dictate which additional steps will be required to remove DNA and protein from the RNA preparation, and whether compartmentalized nuclear RNA and cytoplasmic RNA species can be purified independently of one another.

Alternatively, hnRNA (nuclear RNA) alone can be isolated using this same gentle lysis buffer which, when used correctly, does not cause nuclear breakage. This facilitates the recovery of intact nuclei that can be washed free from any residual cytoplasmic transcripts. 2 Harsh Lysis Buffers There is probably no better way to deal with seemingly recalcitrant RNases than to disrupt cells in a guanidinium lysis buffer [3]. On contact, guanidinium-containing buffers distort the tertiary folding of RNases, which results in their inactivation.

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