Reagent for the isolation of RNA
View Patent ↗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:
about 1% IGEPAL® (tergitol);
about 100 mM EDTA;
about 0.2% SDS;
about 40% 2-mercaptoethanol; and
about 0.5% sodium azide.
2. A reagent for extraction of RNA comprising:
about 1% IGEPAL® (tergitol);
about 100 mM EDTA;
about 0.02% SDS;
about 20% 2-mercaptoethanol; and
about 0.5% sodium azide.
3. A reagent for extraction of RNA comprising:
about 0.5-3% IGEPAL® (tergitol);
about 0.02-0.25 M EDTA;
about 0.01-0.5% SDS;
about 1-40% 2-mercaptoethanol; and
an amount of sodium azide effective to extend the shelf life of the reagent.
4. The reagent of claim 3 , comprising:
about 1-2% IGEPAL® (tergitol).
5. The reagent of claim 3 , comprising:
about 0.01-0.1% SDS.
6. The reagent of claim 3 , comprising:
about 0.02-0.2% SDS.
7. The reagent of claim 3 , comprising:
about 0.5-0.2 M EDTA.
8. The reagent of claim 7 , comprising:
about 0.1 M EDTA.
9. The reagent of claim 3 , comprising:
about 10-40% 2-mercaptoethanol.
10. The reagent of claim 3 , comprising:
about 4-40% 2-mercaptoethanol.
11. The reagent of claim 3 free of phenol.
12. The reagent of claim 3 , comprising: about 0.5% sodium azide.
13. A method for isolating RNA from plant material comprising the following:
mixing the material with the extraction reagent according to claim 3 to form an extract.
14. The method of claim 13 , further comprising the following:
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.
15. The method according to claim 13 , 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.
16. The method according to claim 14 , wherein the cellular debris is removed by centrifugation.
17. The method according to claim 14 , wherein the organically extracting comprises chloroform extraction.
18. The method according to claim 14 , wherein the precipitating comprises alcohol precipitation.
19. A method for isolating RNA from plant material comprising the following:
mixing the material with the extraction reagent according to claim 3 to form an extract; and
separating cellular debris from said extract to form a clarified fraction.
20. The method of claim 19 , further comprising the following:
binding said RNA to a solid matrix.
21. The method according to claim 20 , wherein said binding preferentially binds mRNA.
22. The method according to claim 20 , further comprising eluting said RNA from said solid matrix.
23. A method for isolating RNA from plant material comprising:
exposing a plant material comprising a plant tissue, fungal mycelium or seed to the reagent of claim 3 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.
24. The method according to claim 23 , wherein the separating includes filtering or straining.
25. The method according to claim 23 , wherein the separating includes precipitating RNA and collecting the precipitate.
26. A kit for extracting RNA comprising one or more of the following components:
one or more RNA extraction reagent according to claim 3 ;
one or more RNase free wash reagents;
one or more tissue filters; and
one or more RNase free sample holding tube.
27. The kit according to claim 26 , further comprising:
components for organic extraction of said RNA.
28. The kit according to claim 26 , further comprising:
an RNase free matrix for binding RNA.