Improved Methods for Extracting RNA from Exfoliated Human Colonocytes in Stool and RT-PCR Analysis
Department of Radiation Oncology, Leo W Jenkins Cancer Center, The Brody School of Medicine (BSOM) at East Carolina University (ECU), Greenville, 27834, USA. Digestive Diseases and Sciences
(Impact Factor: 2.61).
11/2004; 49(11-12):1889-98. DOI: 10.1007/s10620-004-9589-9
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.
Available from: Björn L.D.M. Brücher
- "They showed that RNA can be adequately reverse transcribed, making high-quality copy cDNA. This was followed by PCR using colon cancer-specific gene primers (guanylyl cyclase C and PYPAF5 genes).37 "
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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 03/2013; 4(3):281-95. DOI:10.7150/jca.5836 · 3.27 Impact Factor
Available from: Birgitta Agerberth
- "Supernatant was filtered with 0.2 µm filter, lyophilized, and stored at −20 °C until used. Before using in ELISA, lyophilized stool was dissolved in phosphate buffered saline (PBS) in one-third of the starting volume (27). Cathelicidin LL-37 was measured in stool by ELISA as described earlier (28). "
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ABSTRACT: Antimicrobial peptides represent an important component of the innate immune defenses of living organisms, including humans. They are broad-spectrum surface-acting agents secreted by the epithelial cells of the body in response to infection. Recently, L-isoleucine and its analogues have been found to induce antimicrobial peptides. The objectives of the study were to examine if addition of L-isoleucine to oral rehydration salts (ORS) solution would reduce stool output and/or duration of acute diarrhoea in children and induce antimicrobial peptides in intestine. This double-blind randomized controlled trial was conducted at the Dhaka Hospital of ICDDR,B. Fifty male children, aged 6-36 months, with acute diarrhoea and some dehydration, attending the hospital, were included in the study. Twenty-five children received L-isoleucine (2 g/L)-added ORS (study), and 25 received ORS without L-isoleucine (control). Stool weight, ORS intake, and duration of diarrhoea were the primary outcomes. There was a trend in reduction in mean +/- standard deviation (SD) daily stool output (g) of children in the L-isoleucine group from day 2 but it was significant on day 3 (388 +/- 261 vs. 653 +/- 446; the difference between mean [95% confidence interval (CI) (-)265 (-509, -20); p = 0.035]. Although the cumulative stool output from day 1 to day 3 reduced by 26% in the isoleucine group, it was not significant. Also, there was a trend in reduction in the mean +/- SD intake of ORS solution (mL) in the L-isoleucine group but it was significant only on day 1 (410 +/- 169 vs. 564 +/- 301), the difference between mean (95% CI) (-)154 (-288, -18); p = 0.04. The duration (hours) of diarrhoea was similar in both the groups. A gradual increase in stool concentrations of beta-defensin 2 and 3 was noted but they were not significantly different between the groups. L-isoleucine-supplemented ORS might be beneficial in reducing stool output and ORS intake in children with acute watery diarrhoea. A further study is warranted to substantiate the therapeutic effect of L-isoleucine.
Journal of Health Population and Nutrition 06/2011; 29(3):183-90. DOI:10.3329/jhpn.v29i3.7864 · 1.04 Impact Factor
Available from: Paul Vos
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ABSTRACT: There is a need for sensitive and specific diagnostic and prognostic molecular markers which can monitor early patterns of gene expression in non-invasive exfoliated colonocytes shed in the stool, and aggression in carcinoma cells in blood of resected colorectal cancer patients. RNA-based detection methods are more comprehensive than either DNA- or protein-based methods. By routinely and systematically being able to perform quantitative gene expression studies on non-invasive samples using carefully selected tumor-specific colon cancer genes, we can quantitatively and accurately monitor changes at various stages in the neoplastic process, allowing for surgical and/or other therapies, and thus, decrease mortality from colorectal cancer.
Anticancer research 11/2004; 24(6):4127-34. · 1.83 Impact Factor
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