Analyses based on RNA have two major advantages over DNA analyses. First, they are by definition restricted to a step in the expression of proteins from an organism’s genome. This means that RNA pools are generally far less complex than are pools of DNA representative of the organism’s entire genome, and that a quantitative estimate of different RNA’s can provide a useful surrogate for the proteins produced at that time point for a specific organism or tissue within an organism. Second, since nearly all of the recognized viral threats to honey bee exist without a DNA stage, these threats are only visible via RNA analyses. These arguments make RNA the resource of choice for many honey bee analyses; despite greater concerns over storage and preservation of tissues.
A common strategy is to extract total nucleic acids directly in strongly denaturing buffers, so as to inactivate RNAses immediately during homogenisation. RNAses have numerous disulphide bridges. This makes them very stable in a very wide range of conditions, such that strong denaturants are required to permanently inactivate them. Heat, detergents (sodium dodecyl sulphate), organic solvents (phenol), proteinases, chaotropic salts (guanidine isothiocyanate), reducing agents (β-mercaptoethanol; dithiothreitol) and nucleic acid protecting compounds (CTAB; cetyl trimethylammonium bromide) are some of the more common methods used to inactivate RNAses. The nucleic acids can be purified from other compounds with affinity columns, magnetic bead-linked nucleic acid binding agents or by precipitation with alcohol or lithium chloride. The most common, quickest and most reliable combination is a chaotropic salt/β-mercaptoethanol extraction buffer, followed by purification on disposable affinity columns (Verheyden et al., 2003). The main disadvantage of RNA precipitation (with 2 volumes ethanol, 1 volume isopropanol or with 6M LiCl) is that many undesirable compounds often co-precipitate with the nucleic acid, requiring further precipitations or washes to clean the sample. There are many excellent commercial RNA extraction kits available, based on one or more of these principles. However, their performance in comparative tests varies greatly, depending on the organism, tissue type and nucleic acid extracted (Konomi et al., 2002; Knepp et al., 2003; Wilson et al., 2004; Schuurman et al., 2005; Labayru et al., 2005). Below are two protocols, representing the most common approaches to RNA extraction.