Author
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Owens, Robert |
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Submitted to: Current Protocols in Microbiology
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 2/1/2006 Publication Date: 8/1/2008 Citation: Owens, R.A. 2008. Detection of viroids by gel electrophoresis. Current Protocols in Microbiology. 16G.1.1-16G1.9. Interpretive Summary: Viroids are the smallest known agents of infectious disease – small, circular RNA molecules that lack the coat protein characteristic of most conventional viruses yet are able to multiply and cause disease in susceptible host plants. A number of rapid, sensitive, and reliable diagnostic tests have been developed to control viroid diseases, but most such tests require knowledge of the nucleotide sequence of the viroid. Return polyacrylamide gel electrophoresis (R-PAGE) is the only technique for viroid diagnosis that requires no sequence information. Thus, it is very useful for detecting previously unknown viroids. This article provides a step-by-step explanation of how to carry out R-PAGE and is intended for technicians or beginning graduate students with little or no previous experience with electrophoresis techniques. Technical Abstract: Two-dimensional PAGE involving an initial fractionation under non-denaturing conditions followed by a second electrophoresis under denaturing conditions provides a powerful means to detect viroids and other small circular RNAs. This unit describes a method known as “R(eturn) PAGE” in which denaturation is achieved by simultaneously raising the temperature and lowering the ionic strength during the second electrophoresis. Under denaturing conditions, circular RNAs migrate more slowly than the corresponding linear forms. Following fractionation, RNAs are visualized by staining with ethidium bromide, SYBR Gold, or silver nitrate. Unlike nucleic acid hybridization or RT-PCR, viroid identification by R-PAGE requires no nucleotide sequence information. |
