Terbium citrate staining represents the method of choice for RNA visualization at transmission electron microscopy. Because of its affinity for guanosines in RNA rather than in DNA, terbium citrate gives precise results being a selective staining. However, difficulties often arise when performing this technique, especially in crucial and delicate steps such as rinses, when it is not uncommon to excessively reduce the already feeble contrast. For these reasons, we developed a straightforward and secure approach to overcome such complications. Here we report a new method for RNA single molecule localization by means of terbium citrate vapors, viable for every type of fixation and embedding.

The circular and linear forms of viroid RNAs can be separated by two-dimensional polyacrylamide gel electrophoresis (PAGE) based on the selective delay in mobility that circular RNAs experience under denaturing conditions. First PAGE separates RNA preparations from viroid-infected plants, and the whole lane from this first gel is next perpendicularly loaded on top of a second gel. Separation continues under new conditions that differ in the degree of denaturation from the first. The result is a two-dimensional separation of the RNAs in which circular and linear molecules are distributed in two parallel diagonals.

Although the EDTA regressive technique allows the visualization of RNPs, this widely used method is not intended to be specific for RNA alone. A fine ultrastructural visualization of RNA on ultrathin sections can be obtained with terbium citrate: this method gives a weak contrast but a very fine end product which allows observations at a high resolution level.The procedure is very simple since it consists only of a period of incubation and very short washes, which are the crucial point of this technique to avoid the Tb removal from RNA. This method does not require any special type of fixation and embedding.

The number of investigators using cell death analysis applications has greatly expanded since the introduction of flow cytometry. The Annexin V/propidium iodide (PI) method is among the most commonly used procedures and allows users to determine if cells are viable, apoptotic, or necrotic, based on changes in membrane lipid composition, integrity, and permeability. Unfortunately, PI can intercalate into RNA, in addition to DNA, which contributes to a large number of events showing PI staining within the cytoplasmic compartment. We show that this occurs across a broad range of animal primary cells and commonly used cell lines, and is most prevalent in large cells (nuclear:cytoplasmic ratio <0.5). Any cellular system where RNA levels change throughout an experiment will be particularly affected, such as those that utilize virally infected cells. As two examples, we highlight our recent work on cells infected with vesicular stomatitis virus (VSV), an RNA virus, and herpes simplex virus-1 (HSV-1), a DNA virus. Similarly, these issues are relevant to experimental systems where cells have increased RNA content such as during genotoxic stress, following exposure to cell cycle arrest drugs such as thymidine or hydroxyurea, or where developmental progression promotes discrete changes in cellular RNA synthesis. This chapter outlines a modified Annexin V/PI method that addresses cytoplasmic RNA staining issues to allow for accurate assessment of cell death. This protocol takes advantage of an additional cellular permeability caused by fixation to promote RNase A entry into the cell. Based on our observations, cell morphological parameters are well maintained and less than 5 % of total cellular events exhibit cytoplasmic PI staining under this protocol.