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Process and kit for isolating and purifying RNA from biological sources
| Details |
Inventors: Gillespie, David; Cuddy, Kevin K.;
Assignee: Hahnemann University (Philadelphia, PA)
Primary Examiner: Lacey; David L.
Assistant Examiner: Wang; Gian P.
Attorney, Agent or Firm: Dann, Dorfman, Herrell and Skillman
A process is provided for isolating and purifying biologically active RNA from a biological sources containing RNA, DNA and other cellular materials. The process involves contacting the RNA-containing source with particles comprising siliceous material, such as finely-divided glass, in the presence of a binding solution comprising concentrated, acidified chaotropic salt. Under these conditions, RNA, but not DNA, binds selectively to the siliceous material, and can be separated easily from the other components of the sample. Preferably, the selective binding process is applied to biological cells containing RNA of interest. Intact cells are disrupted by exposing them to a lysing solution comprising, as its main component, concentrated, acidified chaotropic salt. The RNA is then isolated and purified from the lysate using the selective binding process of the invention. The process of the invention may be conveniently provided as a kit, which may include the siliceous material, reagents and instructions for use of the kit to isolate and purify biologically active RNA from biological sources. |
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DETAILED DESCRIPTION OF THE INVENTION Sections I-III of the following description detail the preparation and use of a simple, effective method for recovering substantially pure RNA from a mixture of biological substances according to the present invention.
Although the process of the invention is described with reference to contacting a biological source of RNA with glass powder in the presence of a binding solution comprising concentrated, acidified chaotropic salt followed by washing and eluting with specific reagents, various alternative particulate binding substrates and reagents may be used, if desired, as will be further described below.
The selective binding of RNA to the particulate siliceous material under the conditions described herein facilitates its isolation from the other biological substances contained in the RNA source.
For purposes of the present invention, a chaotropic salt is defined as the salt of a chaotropic agent, which is an agent capable of denaturing proteins.
Preferred chaotropic salts for use in the practice of this invention include guanidine thiocyanate, guanidine isothiocyanate and guanidine HCl.
Guanidine thiocyanate and guanidine isothiocyanate are especially preferred for use in the invention because both the guanidinium and the thiocyanate ions act as chaotropic agents.
The inventors recognize that guanidine HCl may not be considered to be a chaotropic salt (e.
g.
, see Gillespie, U.
S.
patent application Ser.
No.
07/299,150, at page 81).
However, acidifed GuHCl acts to promote selective binding of RNA to siliceous material in biological sources previously prepared with guanidine thiocyanate or sodium perchlorate.
In the practice of the present invention, therefore, guanidine HCl is considered to be a chaotrope.
Acidification of a strong chaotropic salt solution enables selective isolation and purification of RNA, rather than co-isolation of DNA and RNA, as was previously done using the glass-binding process described above.
The description which follows sets forth the best mode presently contemplated for practicing the present invention
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Liquid Purification 
