Digestive Diseases and Sciences

, Volume 49, Issue 11–12, pp 1889–1898 | Cite as

Improved Methods for Extracting RNA from Exfoliated Human Colonocytes in Stool and RT-PCR Analysis

  • Farid E. Ahmed
  • Stephanie I. James
  • Donald T. Lysle
  • Larry J. DobbsJr.
  • Roberta M. Johnke
  • Gordon Flake
  • Patricia Stockton
  • Dennis R. Sinar
  • Wade Naziri
  • Mark J. Evans
  • Charles J. Kovacs
  • Ron R. Allison
Article

Abstract

In order to diagnose colon cancer at an earlier, more localized stage, there is a need to develop diagnostic markers (genes) which can detect early patterns of gene expression in exfoliated colonocytes shed in the stool during routine screening for this disease. An RNA-based detection is more pertinent than either a DNA-based or a protein-based method as a screening procedure, but it has not been widely used as a cancer screen because of the difficulty of handling and stabilizing the RNA molecule. We describe a method that permits extraction of intact nondegraded total RNA from human colonocytes in stool and from normal and malignant colon tissues (which were employed for comparison with stool). Because it utilizes commercially available kits, this method is simpler than other published methods and does not require isolation of messenger (m)RNA, thereby reducing the chances of contaminating the preparations with degrading nucleases, and even a small amount of isolated total RNA can be adequately reverse transcribed, making high-quality copy (c) DNA. This is followed by PCR (either qualitative end point or semiquantitative real-time) using colon cancer-specific gene primers. By routinely and systematically being able to perform quantitative gene expression measurements on noninvasive samples, the goal of this pilot work is to lay the groundwork for conducting a large clinical study to identify groups of selected genes whose expression is consistently altered at an early stage in the neoplastic process. Such work will permit noninvasive monitoring of at-risk patients through the analysis of their stool samples. Correct diagnosis will allow for surgical and/or other interventions before the tumor is well established and, thus, should decrease mortality from this preventable disease.

colon cancer DNA diagnosis genes markers RNA screening 

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Copyright information

© Springer Science+Business Media, Inc. 2004

Authors and Affiliations

  • Farid E. Ahmed
    • 1
  • Stephanie I. James
    • 2
  • Donald T. Lysle
    • 3
  • Larry J. DobbsJr.
    • 3
  • Roberta M. Johnke
    • 1
  • Gordon Flake
    • 4
  • Patricia Stockton
    • 4
  • Dennis R. Sinar
    • 5
  • Wade Naziri
    • 6
  • Mark J. Evans
    • 1
  • Charles J. Kovacs
    • 1
  • Ron R. Allison
    • 1
  1. 1.Department of Radiation Oncology, Leo W. Jenkins Cancer CenterThe Brody School of Medicine (BSOM) at East Carolina University (ECU)Greenville
  2. 2.Department of PsychologyUniversity of North CarolinaChapel Hill
  3. 3.Department of Pathology and Laboratory MedicineThe BSOM at ECUGreenville
  4. 4.Laboratory of Experimental PathologyNational Institute of Environmental Health Sciences (NIEHS)Research Triangle Park
  5. 5.Department of Internal MedicineThe BSOM at ECUGreenville
  6. 6.Carolina Physicians PAGreenvilleUSA

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