Invasive detection of colonic biomarkers
OwnerRobert S. Chapkin, Laurie A. Davidson +1inventors
InventorsRobert S. Chapkin, Laurie A. Davidson, Joanne R. Lupton
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Abstract
A noninvasive method utilizing feces, containing sloughed colonocytes, as a sensitive technique for detecting diagnostic colonic biomarkers as well as a method for isolating poly A+ RNA from feces. The method allows the isolation and quantitation of specific eukarotic mRNAs as candidate biomarkers for colon cancer isolated from feces.
Claims33
A method for non-invasively determining the expression of PKC isozymes in colonocytes of a patient comprising:
- directly isolating from a patient poly A+ RNA from feces, containing sloughed colonocytes;
- and
- assaying the isolated poly A+ RNA and determining the level, in the isolated A+ RNA, of mRNA encoding at least one PKC isozyme.
A method for non-invasively detecting colonic biomarkers in a patient using fecal messenger RNA comprising:
- directly isolating, from said patient, poly A+ RNA from feces containing sloughed colonocytes;
- and
- assaying the isolated poly A+ RNA and determining the level, in the isolated poly A+ RNA, of mRNA encoding at least one colonic biomarker.
A method for non-invasively screening for colon cancer in a patient comprising:
- detecting the expression of at least one specific biomarker in sloughed coloncytes in said patient's feces;
- and
- correlating the expression of said at least one specific biomarker with the presence or absence of colon cancer in said patient.
A method for isolating poly A+ RNA from feces comprising the steps of:
- homogenizing feces;
- adding a dilution buffer to the feces to form a fecal homogenate;
- centrifuging the fecal homogenate to create a supernatant;
- adding oligo dT cellulose to the supernatant to form an oligo dT cellulose supernatant mixture;
- mixing the oligo dT cellulose supernatant mixture;
- centrifuging the oligo dT cellulose supernatant mixture to form a pellet;
- resuspending the pelleted resin with a binding buffer;
- centrifuging the resuspended resin to form a pellet and discarding the supernatant;
- resuspending the pelleted resin with a wash buffer;
- centrifuging the resuspended resin to form a pellet and discarding the supernatant;
- resuspending the pelleted resin with an elution buffer;
- eluting the poly A+ RNA by centrifugation;
- precipitating the poly A+ RNA by adding a precipitation solution and chilling the resuspension;
- and
- centrifuging the chilled resuspension to pellet poly A+ RNA.
Dependencies are inferred from the claim text.