Article

Digestive Diseases and Sciences

, Volume 49, Issue 11, pp 1889-1898

First online:

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

  • Farid E. AhmedAffiliated withDepartment of Radiation Oncology, Leo W. Jenkins Cancer Center, The Brody School of Medicine (BSOM) at East Carolina University (ECU) Email author 
  • , Stephanie I. JamesAffiliated withDepartment of Psychology, University of North Carolina
  • , Donald T. LysleAffiliated withDepartment of Pathology and Laboratory Medicine, The BSOM at ECU
  • , Larry J. DobbsJr.Affiliated withDepartment of Pathology and Laboratory Medicine, The BSOM at ECU
  • , Roberta M. JohnkeAffiliated withDepartment of Radiation Oncology, Leo W. Jenkins Cancer Center, The Brody School of Medicine (BSOM) at East Carolina University (ECU)
  • , Gordon FlakeAffiliated withLaboratory of Experimental Pathology, National Institute of Environmental Health Sciences (NIEHS)
  • , Patricia StocktonAffiliated withLaboratory of Experimental Pathology, National Institute of Environmental Health Sciences (NIEHS)
  • , Dennis R. SinarAffiliated withDepartment of Internal Medicine, The BSOM at ECU
  • , Wade NaziriAffiliated withCarolina Physicians PA
    • , Mark J. EvansAffiliated withDepartment of Radiation Oncology, Leo W. Jenkins Cancer Center, The Brody School of Medicine (BSOM) at East Carolina University (ECU)
    • , Charles J. KovacsAffiliated withDepartment of Radiation Oncology, Leo W. Jenkins Cancer Center, The Brody School of Medicine (BSOM) at East Carolina University (ECU)
    • , Ron R. AllisonAffiliated withDepartment of Radiation Oncology, Leo W. Jenkins Cancer Center, The Brody School of Medicine (BSOM) at East Carolina University (ECU)

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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