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Purification of total RNA from human stool samples
1Department of Veterans Affairs Medical Center, New York, New York 10010, USA.
Digestive Diseases and Sciences
|January 9, 1999
Summary
Researchers developed a novel method to isolate RNA from human stool, enabling the analysis of gene expression in shed cells. This technique may offer a less invasive approach for early colon cancer detection.
Area of Science:
- Molecular Biology
- Gastroenterology
- Biochemistry
Background:
- Colonoscopy is effective for early colon tumor detection but is invasive.
- A less invasive and more cost-effective method for mucosal sampling is needed.
- Stool contains sloughed-off colonic cells, potentially carrying tumor-specific gene expression alterations.
Purpose of the Study:
- To develop a method for isolating total RNA from human stool.
- To confirm the presence of human gene transcripts in isolated stool RNA.
- To assess the potential of this method for colon cancer risk assessment.
Main Methods:
- Developed a stool RNA isolation protocol involving lysis, phenol extraction, and RNA-binding resin.
- Treated isolated RNA with RNase-free DNase I.
- Assayed for human RNA using quantitative slot blotting, northern blotting, and RT-PCR.
- Validated RNA isolation by detecting rat-specific mRNA in a mixed stool sample.
Main Results:
- Successfully isolated 5-30 microg RNA per gram of stool from cancer patients and 5 microg/g from controls.
- Quantitative slot blotting indicated approximately 10% of isolated RNA was of human origin.
- Northern blotting and RT-PCR confirmed the presence of human RNA transcripts.
- RT-PCR unambiguously detected rat-specific mRNA, validating the isolation method.
Conclusions:
- RNA can be successfully isolated from human stool.
- Human gene transcripts are detectable in stool-derived RNA preparations.
- This RNA isolation technique offers a promising, non-invasive tool for identifying individuals at risk for colon cancer.