Reagent for the isolation of RNA

Abstract

The present invention provides RNA extraction reagents, methods and kits that are especially useful for extracting RNA. The reagents, methods and kits of the present invention are especially useful for extracting RNA, for example, cytoplasmic RNA, from difficult materials, from plants, especially, difficult plant tissues, such as those containing phenolics, tannins, polysaccharides (such as starch) and resins. Comparative high yields are obtainable according to the present invention when compared to conventional reagents and methods. The RNA preparations obtained in accordance with the present invention are also of high quality as demonstrated by superior A 260/280 results and by gel electrophoresis.

1 . A reagent for extraction of RNA comprising one or more of the following components: 
 at least one non-ionic detergent;    at least one ionic detergent;    at least one chelator; and    at least one reducing agent.    
     
     
         2 . The reagent according to  claim 1 , further comprising an antibacterial agent (e.g., sodium azide, 0.5%)  
     
     
         3 . The reagent according to  claim 1 , wherein the non-ionic detergent comprises a tert-octylphenoxy poly(oxyethylene)ethanol.  
     
     
         4 . The reagent according to  claim 1 , wherein the ionic detergent comprises SDS.  
     
     
         5 . The reagent according to  claim 1 , wherein the chelator comprises EDTA or EGTA.  
     
     
         6 . The reagent according to  claim 1 , wherein the reducing agent comprises 2-mercaptoethanol or dithiothreitol.  
     
     
         7 . The reagent according to  claim 1  comprising: 
 a tert-octylphenoxy poly(oxyethylene)ethanol;    SDS;    EDTA; and    2-mercaptoethanol or dithiothreitol.    
     
     
         8 . The reagent according to  claim 1  comprising: 
 at least one non-ionic detergent at a concentration of 0.1-4% by volume;    at least one ionic detergent at a concentration of 0-1% by weight;    at least one chelator at a concentration of 0.02-0.25 M; and    at least one reducing agent at a concentration of 1-40% by volume.    
     
     
         9 . The reagent according to  claim 8  comprising: 
 about 1% Igepal;    about 100 mM EDTA;    about 0.2% SDS;    about 40% 2-mercaptoethanol; and    about 0.5% sodium azide.    
     
     
         10 . The reagent according to  claim 8  comprising: 
 about 1% Igepal;    about 100 mM EDTA;    about 0.02% SDS;    about 20% 2-mercaptoethanol; and    about 0.5% sodium azide.    
     
     
         11 . A method for isolating RNA from plant material comprising one or more of the following: 
 mixing the material with the extraction reagent according to  claim 1  to form an extract;    separating cellular debris from said extract to form a clarified fraction;    organically extracting said clarified fraction to form an aqueous phase and an organic phase; and    precipitating RNA from said aqueous phase.    
     
     
         12 . The method according to  claim 11 , wherein the plant material comprises plant tissue, fungal mycelium or seed, said method further comprising pulverizing the tissue or seed to form a powder or paste.  
     
     
         13 . The method according to  claim 11 , wherein the cellular debris is removed by centrifugation.  
     
     
         14 . The method according to  claim 11 , wherein the organically extracting comprises chloroform extraction.  
     
     
         15 . The method according to  claim 11 , wherein the precipitating comprises alcohol precipitation.  
     
     
         16 . A method for isolating RNA from plant material comprising one or more of the following: 
 mixing the material with the extraction reagent according to  claim 1  to form an extract;    separating cellular debris from said extract to form a clarified fraction; and    binding said RNA to a solid matrix.    
     
     
         17 . The method according to  claim 16 , wherein said binding preferentially binds mRNA.  
     
     
         18 . The method according to  claim 16 , further comprising eluting said RNA form said solid matrix.  
     
     
         19 . A method for isolating RNA from plant material comprising: 
 exposing a plant material comprising a plant tissue, fungal mycelium or seed to a permeabilizing reagent to permit cytoplasmic RNA to extract from cells or cell debris of said plant material; and    separating said cytoplasmic RNA from said cells or cellular debris.    
     
     
         20 . The method according to  claim 18 , wherein the separating includes filtering or straining.  
     
     
         21 . The method according to  claim 18 , wherein the separating includes precipitating RNA and collecting the precipitate.  
     
     
         22 . A kit for extracting RNA comprising one or more of the following components: 
 one or more RNA extraction reagent according to  claim 1;     one or more RNase free wash reagents;    one or more tissue filters; and    one or more RNase free sample holding tube.    
     
     
         23 . The kit according to  claim 22 , further comprising: 
 components for organic extraction of said RNA.    
     
     
         24 . The kit according to  claim 22 , further comprising: 
 an RNase free matrix for binding RNA.