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Chapter 44 : Exploiting MicroRNA (miRNA) Profiles for Diagnostics

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Exploiting MicroRNA (miRNA) Profiles for Diagnostics, Page 1 of 2

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Abstract:

Clinical management of any disease depends upon timely, accurate, and sensitive diagnosis of the etiology of disease to determine an appropriate counter strategy. As defined by the Biomarkers Definitions Working Group, biomarkers are characteristics that are objectively measured and evaluated as indicators of normal biological, pathological, or pharmacological responses to a disease or therapeutic intervention (1, 2). An ideal biomarker should be inexpensive to detect; readily assayed; present at favorable concentrations in cells, target tissues, and/or in biological fluids; and resistant to degradation during typical storage. The biomarker should also provide insights into disease etiology, progression, and/or treatment efficacy. Biomarkers can indicate toxicity, safety, efficacy, pharmacodynamics, disease diagnosis, or prognosis following treatment or at clinical endpoints (3). Assessment of molecular biomarkers has been typically slow, expensive, and time consuming (4–6). Factors that contribute to this are the collection methods and the need for preservation, purification, and environmental stability of biological samples. Three large consortia, the NCI Early Detection Research Network (EDRN), Critical Path Predictive Safety Testing Consortium (PSTC), and the Alzheimer's Disease Neuroimaging Initiative (ADNI), are presently involved in screening thousands of biomolecules as potential biomarkers. Irrespective of their role, discovering biomarkers relies on defining their intended roles (diagnosis/prognosis, drug efficacy, sample type, assay to be used, etc.) in day-to-day clinical practice.

Citation: Bakre A, Tripp R. 2016. Exploiting MicroRNA (miRNA) Profiles for Diagnostics, p 634-654. In Persing D, Tenover F, Hayden R, Ieven M, Miller M, Nolte F, Tang Y, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555819071.ch44
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Figures

Image of FIGURE 1
FIGURE 1

miRNA biogenesis pathways in animal cells. miRNA genes encoded in intronic/intergenic regions of the genome are transcribed by RNA polymerase II and processed by nuclear RNase III Drosha to generate pre-miRNAs that are actively exported out of the nucleus into the cytosol. Pre-miRNAs are further processed by a second RNase III Dicer to generate the mature miRNA dsRNA duplex that associates with Argonaute and other proteins to form the miRISC complex where posttranscriptional inhibition initiates.

Citation: Bakre A, Tripp R. 2016. Exploiting MicroRNA (miRNA) Profiles for Diagnostics, p 634-654. In Persing D, Tenover F, Hayden R, Ieven M, Miller M, Nolte F, Tang Y, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555819071.ch44
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Image of FIGURE 2
FIGURE 2

The role of miRNAs in the regulation of the host immune response. (A) Major pathways involved in the innate immune pathway are shown with major adaptor molecules. miRNAs that regulate these genes are shown in red. (B) The role of miRNAs in B- and T-cell development and function is summarized.

Citation: Bakre A, Tripp R. 2016. Exploiting MicroRNA (miRNA) Profiles for Diagnostics, p 634-654. In Persing D, Tenover F, Hayden R, Ieven M, Miller M, Nolte F, Tang Y, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555819071.ch44
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Image of FIGURE 3
FIGURE 3

Overview of electrochemical and direction detection methods for miRNAs. (A) miRNAs bind to fluorophore-labeled probe in solution and are captured by paramagnetic beads coated with p19 protein which binds only dsRNAs. Bound hybrids are resolved by capillary electrophoresis alone or in the presence of buffer alone or single-strand-binding protein. Time resolved fluorescence intensity determines miRNA abundance in sample. (B) miRNAs are captured on surface-bound DNA probes and then cleaved by double-strand specific nuclease that changes the electrochemical signal on the chip in a concentration-dependent manner. (C) A triplex sensor-based approach based on hybridization; p19 binding and p19 displacement detect changes in square wave voltages in a miRNA concentration-dependent fashion. (D) miRNA binding to a p19 array on a carbon nanotube array causes reduction in current flow through the chip in a miRNA concentration-dependent manner. (E) Porous hydrogel-based microbeads carry miRNA probes which bind to target miRNAs. Biotin-adaptor oligos are attached to the hybrid followed by binding of a streptavidin-PE label and flow cytometry of the sample to detect miRNAs. (F) Molecular beacons bind to miRNA via a complementary region leading to spatial separation of the fluorophore and quencher on the beacon and production of a fluorescent signal.

Citation: Bakre A, Tripp R. 2016. Exploiting MicroRNA (miRNA) Profiles for Diagnostics, p 634-654. In Persing D, Tenover F, Hayden R, Ieven M, Miller M, Nolte F, Tang Y, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555819071.ch44
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Image of FIGURE 4
FIGURE 4

Overview of RNA isolation strategies for assessing miRNAs in cells/tissue or clinical samples. Tissues/biofluids are lysed in a lysis buffer, fractionated, and then eluted from silica columns or precipitated using a salt + alcohol combination. Size-fractionated RNA can also be isolated using modifications of these protocols.

Citation: Bakre A, Tripp R. 2016. Exploiting MicroRNA (miRNA) Profiles for Diagnostics, p 634-654. In Persing D, Tenover F, Hayden R, Ieven M, Miller M, Nolte F, Tang Y, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555819071.ch44
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Image of FIGURE 5
FIGURE 5

Overview of real-time miRNA detection techniques. Total or size-fractionated RNA is polyadenylated followed by reverse transcription using an adaptor oligo or oligo dT primer. (A) First strand cDNA synthesized can be then PCR amplified using a miRNA-specific forward oligo and a universal PCR oligo (in case of SYBR green chemistry) or a forward oligo, probe, and reverse oligo (in case of TaqMan chemistry) or using molecular beacons. (B) PCR master mix containing first-strand cDNA and miRNA-specific primers/probes is fractionated into nanoliter droplets followed by routine PCR amplification. Amplified product is then analyzed by a modified flow cytometer to detect sample populations.

Citation: Bakre A, Tripp R. 2016. Exploiting MicroRNA (miRNA) Profiles for Diagnostics, p 634-654. In Persing D, Tenover F, Hayden R, Ieven M, Miller M, Nolte F, Tang Y, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555819071.ch44
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