What Protocol modifications should be used for rna Isolation from difficult Tissues? RNA isolation from some tissues requires protocol modifica tions to eliminate specific contaminants, or tissue treatments prior to the rna isolation protocol. Fibrous tissues and tissue rich in protein, DNA and RNases, present unique challenges for total RNA isolation. In this section we address problems presented b difficult tissues and offer troubleshooting techniques to help over- come these problems. A separate section will discuss the homog enization needs of various sample types in greater detail Websitesthatdiscusssimilarissuesarehttp://www.nwfsc. noaa.gov/protocols/methods/rnamethodsmenu.htmlandhttp:// grimwade. biochem. unimelb. edu. au/sigtrans html Fibrous Tissue Good yields and quality of total RNA from fibrous tissue such as heart and muscle are dependent on the complete disruption of the starting material when preparing homogenates. Due to low cell density and the polynucleate nature of muscle tissue, yields are typically low; hence it is critical to make the most of the tissue at hand. Pulverizing the frozen tissue into a powder while keeping the tissue completely frozen(use a chilled mortar and pestle) is the key to isolating intact total RNA. It is critical that there be no discernible lumps of tissue remaining after homogenization Lipid and Polysaccharide-Rich Tissue Plant and brain tissues are typically rich in lipids, which makes it difficult to get clean separation of the RNa and the rest of the cellular debris. When using phenol-based methods to isolate total RNA, white flocculent material present throughout the aqueous phase is a classic indicator of this problem. This flocculate will not accumulate at the interface even after extended centrifuga tion Chloroform: isoamyl alcohol(24: 1)extraction of the lysate is probably the best way to partition the lipids away from the RNA. To minimize loss, back-extract the organic phase, and then clean up the recovered aqueous Rna by extraction with phenol: chloroform: isoamyl alcohol (25: 24: 1) When isolating total RNA from plant tissue using a non-phenol based method, polyvinylpyrrolidone-40(PVP-40)can be added to the lysate to absorb polysaccharide and polyphenolic contami nants. When the lysate is centrifuged to remove cell debris, thes contaminants will be pelleted with the PVP(Fang, Hammar, and Grumet, 1992; see also the chapter by Wilkins and Smart, " Isola tion of RNA from Plant Tissue, " in Krieg, 1996, for a list of refer RNA Purification 207What Protocol Modifications Should Be Used for RNA Isolation from Difficult Tissues? RNA isolation from some tissues requires protocol modifica￾tions to eliminate specific contaminants, or tissue treatments prior to the RNA isolation protocol. Fibrous tissues and tissue rich in protein, DNA and RNases, present unique challenges for total RNA isolation. In this section we address problems presented by difficult tissues and offer troubleshooting techniques to help over￾come these problems. A separate section will discuss the homog￾enization needs of various sample types in greater detail. Web sites that discuss similar issues are http://www.nwfsc. noaa.gov/protocols/methods/RNAMethodsMenu.html and http:// grimwade.biochem.unimelb.edu.au/sigtrans.html. Fibrous Tissue Good yields and quality of total RNA from fibrous tissue such as heart and muscle are dependent on the complete disruption of the starting material when preparing homogenates. Due to low cell density and the polynucleate nature of muscle tissue, yields are typically low; hence it is critical to make the most of the tissue at hand. Pulverizing the frozen tissue into a powder while keeping the tissue completely frozen (use a chilled mortar and pestle) is the key to isolating intact total RNA. It is critical that there be no discernible lumps of tissue remaining after homogenization. Lipid and Polysaccharide–Rich Tissue Plant and brain tissues are typically rich in lipids, which makes it difficult to get clean separation of the RNA and the rest of the cellular debris. When using phenol-based methods to isolate total RNA, white flocculent material present throughout the aqueous phase is a classic indicator of this problem. This flocculate will not accumulate at the interface even after extended centrifuga￾tion. Chloroform :isoamyl alcohol (24 : 1) extraction of the lysate is probably the best way to partition the lipids away from the RNA. To minimize loss, back-extract the organic phase, and then clean up the recovered aqueous RNA by extraction with phenol :chloroform: isoamyl alcohol (25: 24: 1). When isolating total RNA from plant tissue using a non-phenol￾based method, polyvinylpyrrolidone-40 (PVP-40) can be added to the lysate to absorb polysaccharide and polyphenolic contami￾nants. When the lysate is centrifuged to remove cell debris, these contaminants will be pelleted with the PVP (Fang, Hammar, and Grumet, 1992; see also the chapter by Wilkins and Smart, “Isola￾tion of RNA from Plant Tissue,” in Krieg, 1996, for a list of refer￾RNA Purification 207