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Maximize Your RNA Yield — What Yield to Expect
RNA content can vary widely between tissues, cell-types, physiological state, etc. If you are accustomed to working with tissues where RNA is plentiful, such as liver, you may have unrealistically high expectations of RNA yields from tissues with lower RNA contents (e.g. skin, muscle, bone). Tables 1 and 2 provide general guidelines for estimating RNA yields from a variety of cells and tissues. Remember that RNA yields from analogous tissues in different organisms can also vary.
Table 1. Total RNA Yields from 1 mg of Various Tissues.
Table 2. General Guideline for the Amount of Total and Poly(A) RNA to Expect from Tissues and Cells.
Cellular Disruption — Get the RNA Out
Thorough cellular disruption is critical for high RNA quality and yield. RNA that is trapped in intact cells is often removed with cellular debris and is unavailable for subsequent isolation. Therefore, it is crucial to choose the disruption method best suited to your tissue or organism to maximize yield. Table 3 summarizes our recommendations for cellular disruption for different specimens. Mechanical cell disruption techniques include grinding, homogenization with dounce or rotor-stator homogenizers (polytron), vortexing, sonication, and use of bead and freezer mills. Complete disruption of some tissues may require using a combination of these techniques. Rotor-stator homogenizers, alone or in conjunction with other disruption techniques, generally result in higher RNA yields than other types of homogenizers.
Table 3. Recommendation for Cellular Disruption of Different Sample Types.
Enzymatic digestion is often recommended for yeast and bacteria to dissolve cell wall structures that are not easily sheared by mechanical forces alone.
Table 3. Recommendation for Cellular Disruption of Different Sample Types.
Enzymatic digestion is often recommended for yeast and bacteria to dissolve cell wall structures that are not easily sheared by mechanical forces alone.
Fine Tuning RNA Extraction to Maximize Yield
Protein-, lipid-, or nucleic acid-rich tissues can present special challenges to both phenol-based and glass-binding, column-based RNA isolation procedures. These tissues may require more manipulation and fine-tuning of RNA isolation procedures to maximize yield and quality.
In phenol-based isolations, such as ToTALLY RNA™ and TRI Reagent® (Ambion's single-step extraction reagent), lysis in a chaotropic reagent is followed by organic extraction. The most frequent problems with phenol-based procedures are incomplete phase separation and/or excessive loss of material at the interface. Dilution of lysates prior to extraction will reduce both the viscosity and the concentration of proteins, lipids, and nucleic acids, although protein- or DNA-rich tissues may require additional phenol:chloroform:IAA extractions to remove these contaminants. Extraction of lipid-rich tissues often results in the formation of a flocculent white precipitate in the aqueous phase. This is likely due to precipitation of insoluble lipids and can be remedied by adding additional chloroform and re-extracting (or chloroform extracting the cell lysate prior to phenol extraction or glass binding). Another manipulation that may maximize RNA recovery from difficult samples is a rapid interface re-spin or back-extraction. To back-extract, the last portion of the aqueous phase and contaminating interface material are transferred to a 1.5 ml tube, the aqueous phase is diluted with more lysis buffer or water, and this mixture is then centrifuged to separate the phases. The clarified aqueous portion can be recovered and pooled with the rest of the aqueous phase to improve RNA recovery. To assure high purity, phenol:chloroform:IAA extractions should be performed until contaminants at the interface are absent.
Column-based procedures, such as RNAqueous™, utilize glass-fiber filters that bind RNA in the presence of chaotropic salts. Proteins and DNA are removed by washing the filter and RNA is then eluted in RNase-free water. The most frequent cause of low RNA yield is overloading the column, which can cause the column to clog or can prevent the RNA from binding efficiently. Methods that reduce viscosity, such as dilution with lysis buffer, extensive mechanical disruption, and centrifugation, will increase RNA yield. If yields are still lower than expected, consider diluting the clarified lysate and splitting it between two columns, which will further reduce the concentration of contaminants and improve RNA binding and recovery. Ambion also offers the Plant RNA Isolation Aid, which can be added to the RNAqueous Lysis/Binding solution prior to homogenization to bind and remove polysaccharides and polyphenols commonly present in plant tissues.
In phenol-based isolations, such as ToTALLY RNA™ and TRI Reagent® (Ambion's single-step extraction reagent), lysis in a chaotropic reagent is followed by organic extraction. The most frequent problems with phenol-based procedures are incomplete phase separation and/or excessive loss of material at the interface. Dilution of lysates prior to extraction will reduce both the viscosity and the concentration of proteins, lipids, and nucleic acids, although protein- or DNA-rich tissues may require additional phenol:chloroform:IAA extractions to remove these contaminants. Extraction of lipid-rich tissues often results in the formation of a flocculent white precipitate in the aqueous phase. This is likely due to precipitation of insoluble lipids and can be remedied by adding additional chloroform and re-extracting (or chloroform extracting the cell lysate prior to phenol extraction or glass binding). Another manipulation that may maximize RNA recovery from difficult samples is a rapid interface re-spin or back-extraction. To back-extract, the last portion of the aqueous phase and contaminating interface material are transferred to a 1.5 ml tube, the aqueous phase is diluted with more lysis buffer or water, and this mixture is then centrifuged to separate the phases. The clarified aqueous portion can be recovered and pooled with the rest of the aqueous phase to improve RNA recovery. To assure high purity, phenol:chloroform:IAA extractions should be performed until contaminants at the interface are absent.
Column-based procedures, such as RNAqueous™, utilize glass-fiber filters that bind RNA in the presence of chaotropic salts. Proteins and DNA are removed by washing the filter and RNA is then eluted in RNase-free water. The most frequent cause of low RNA yield is overloading the column, which can cause the column to clog or can prevent the RNA from binding efficiently. Methods that reduce viscosity, such as dilution with lysis buffer, extensive mechanical disruption, and centrifugation, will increase RNA yield. If yields are still lower than expected, consider diluting the clarified lysate and splitting it between two columns, which will further reduce the concentration of contaminants and improve RNA binding and recovery. Ambion also offers the Plant RNA Isolation Aid, which can be added to the RNAqueous Lysis/Binding solution prior to homogenization to bind and remove polysaccharides and polyphenols commonly present in plant tissues.