Improved methods for extracting RNA from exfoliated human colonocytes in stool and RT-PCR analysis.
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.
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ABSTRACT: Early screening for colon cancer (CC) allows for early stage diagnosis of the malignancy and potentially reduces disease mortality as the cancer is most likely curable at its earliest stages. Early detection would be desirable if accurate, practical and cost-effective diagnostic measures for this cancer were available. Mortality and morbidity from CC represent a major health problem involving a malignant disease that is theoretically preventable through screening. Current screening methods (e.g., the convenient and inexpensive immunological fecal occult blood test, FOBTi, obtained from patients' medical records) either lack sensitivity and require dietary restriction, which impedes compliance and use; are costly (e.g., colonoscopy), which decreases compliance; or could result in mortality. In comparison with the FOBT test, a non-invasive sensitive screen for which there is no requirement for dietary restriction would be a more convenient test. Colorectal cancer is the only cancer for which colonoscopy is recommended as a screening method. Although colonoscopy is a reliable screening tool, the invasive nature, abdominal pain, potential complications and high cost have hampered the application of this procedure worldwide. A screening approach using the stable miRNA molecules, which are relatively non-degradable when extracted from non-invasive stool and semi-invasive blood samples by commercially available kits and manipulated thereafter, would be preferable to a transcriptomic mRNA-, a mutation DNA-, an epigenetic- or a proteomic-based test. The approach uses reverse transcriptase, modified real-time quantitative PCR. Although exosomal RNA would be missed, using a restricted extraction of total RNA from stool or blood, a parallel test could also be carried out on RNA obtained from stool or plasma samples, and appropriate corrections for exsosomal loss can be made for accurate and quantitative test result. Eventually, a chip can be developed to facilitate diagnosis, as has been done for the quantification of genetically modified organisms in foods. The gold standard to which the molecular miRNA test is compared is colonoscopy, which can be obtained from patients' medical records. If performance criteria are met, as detailed herein, a miRNA test in human stool or blood samples based on high-throughput automated technologies and quantitative expression measurements commonly used in the diagnostic clinical laboratory should be advanced to the clinical setting, which will make a significant impact on CC prevention.Expert Review of Anti-infective Therapy 03/2014; · 3.06 Impact Factor
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ABSTRACT: The discovery of microRNA, a group of regulatory short RNA fragments, has added a new dimension to the diagnosis and management of neoplastic diseases. Differential expression of microRNA in a unique pattern in a wide range of tumor types enables researches to develop a microRNA-based assay for source identification of metastatic disease of unknown origin. This is just one example of many microRNA-based cancer diagnostic and prognostic assays in various phases of clinical research.Since colorectal cancer (CRC) is a phenotypic expression of multiple molecular pathways including chromosomal instability (CIN), micro-satellite instability (MIS) and CpG islands promoter hypermethylation (CIMP), there is no one-unique pattern of microRNA expression expected in this disease and indeed, there are multiple reports published, describing different patterns of microRNA expression in CRC.The scope of this manuscript is to provide a comprehensive review of the scientific literature describing the dysregulation of and the potential role for microRNA in the management of CRC. A Pubmed search was conducted using the following MeSH terms, "microRNA" and "colorectal cancer". Of the 493 publications screened, there were 57 papers describing dysregulation of microRNA in CRC.Journal of Cancer. 01/2013; 4(3):281-95.
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ABSTRACT: Colorectal cancer is one of the most common forms of cancer worldwide. Early detection would allow patients to be treated surgically and halt the progression of the disease; however, the current methods of early detection are invasive (colonoscopy and sigmoidoscopy) or have low sensitivity (fecal occult blood test). The altered expression of genes in stool samples of patients with colorectal cancer can be determined by RT-PCR. This is a noninvasive and highly sensitive technique for colorectal cancer screening. According to information gathered in this review and our own experience, the use of fecal RNA to determine early alterations in gene expression due to malignancy appears to be a promising alternative to the current detection methods and owing to its low cost could be implemented in public health services.The International journal of biological markers 03/2012; 27(2):e82-9. · 1.36 Impact Factor