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Colorectal Cancer pp 203–215Cite as

RT-qPCR for Fecal Mature MicroRNA Quantification and Validation

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1765))

Abstract

By routinely and systematically being able to perform quantitative stem-loop reverse transcriptase (RT) followed by TaqMan® minor-groove binding (MGB) probe, real-time quantitative PCR analysis on exfoliated enriched colonocytes in stool, using human (Homo sapiens, hsa) micro(mi)RNAs to monitor changes of their expression at various stages of colorectal (CRC) progression, this method allows for the reliable and quantitative diagnostic screening of colon cancer (CC). Although the expression of some miRNA genes tested in tissue shows less variability in normal or cancerous patients than in stool, the noninvasive stool by itself is well suited for CC screening. An miRNA approach using stool promises to offer more sensitivity and specificity than currently used genomic, methylomic, or proteomic methods for CC screening.

To present an application of employing miRNAs as diagnostic markers for CC screening, we carried out global microarray expression studies on stool colonocytes isolated by paramagnetic beads, using Affymetrix GeneChip miRNA 3.0 Array, to select a panel of miRNAs for subsequent focused semiquantitative PCR analysis studies. We then conducted a stem-loop RT-TaqMan® MGB probes, followed by a modified real-time qPCR expression study on 20 selected miRNAs for subsequent validation of the extracted immunocaptured total small RNA isolated from stool colonocytes. Results showed 12 miRNAs (miR-7, miR-17, miR-20a, miR-21, miR-92a, miR-96, miR-106a, miR-134, miR-183, miR-196a, miR-199a-3p, and miR214) to have an increased expression in stool of CC patients, and that later TNM stages exhibited more increased expressions than adenomas, while 8 miRNAs (miR-9, miR-29b, miR-127-5p, miR-138, miR-143, miR-146a, miR-222, and miR-938) showed decreased expressions in stool of CC patients, which becomes more pronounced as the cancer progresses from early to late TNM stages (0–IV).

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Author information

Authors and Affiliations

  1. GEM Tox Labs, Institute for Research in Biotechnology, Greenville, NC, USA

    Farid E. Ahmed & Nancy C. Ahmed

  2. Department of Nutrition & Food Science, National Research Center, Cairo, Egypt

    Mostafa M. Gouda

  3. Department of Biostatistics, East Carolina University, Greenville, NC, USA

    Paul W. Vos

  4. Department of Physics, East Carolina University, Greenville, NC, USA

    Chris Bonnerup

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  1. Farid E. Ahmed
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  2. Nancy C. Ahmed
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  3. Mostafa M. Gouda
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  4. Paul W. Vos
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  5. Chris Bonnerup
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Editor information

Editors and Affiliations

  1. Laboratory of Intestinal Physiopathology, Department of Anatomy and Cell Biology, Faculty of Medicine and Health Science, Université de Sherbrooke, Sherbrooke, Québec, Canada

    Jean-François Beaulieu

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Ahmed, F.E., Ahmed, N.C., Gouda, M.M., Vos, P.W., Bonnerup, C. (2018). RT-qPCR for Fecal Mature MicroRNA Quantification and Validation. In: Beaulieu, JF. (eds) Colorectal Cancer. Methods in Molecular Biology, vol 1765. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7765-9_13

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  • DOI: https://doi.org/10.1007/978-1-4939-7765-9_13

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7764-2

  • Online ISBN: 978-1-4939-7765-9

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