enzyme, which recognizes a different sequence from the first in order to recirculate
the DNA of interest linked to its interacting DNA. 4C technology is an advanced
3C-based approach, as it combines 3C with NGS technology. A further variant of 3C
is 5C technology, which has the added benefit of studying millions of interactions in
genomic areas by using universal primers in one single assay. 3C-based technologies
include Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIAPET), which combines the 3C method with ChIP and examines genomic sites
associated with specific transcription factors, which can be proximal and distal
[60]. In ChIA-PET, a specific antibody is used together with sonicated chromatin
and the protein of interest, and only this immunoprecipitated material linked to the
protein of interest is used to construct the ChIA-PET. Then, biotinylated oligonucleotides are used to ligate DNA proximal fragments, which are subsequently ligated
on beads. Finally, DNA is digested by a specific restriction enzyme which cuts to
release a tag-linker-tag region. After pull-down of biotinylated linkers by specific
beads, DNA tags are amplified. Hi-ChIP is an advanced Chia-PET method [61]
which provides a more accurate interpretation of information on genome conformation. In Hi-ChIP, DNA interactions are firstly established in situ in the nucleus before
lysis procedure. In this way it is possible to avoid false-positive interactions.
3 Epigenetic Regulatory Role of miRNA: New Analytical
Tools and Technologies
miRNAs are small non-coding RNAs whose role as actors and targets in epigenetic machinery is well established. They are efficient regulators of heterogeneous
physiological and pathological functions, as is well described in almost every
cancer model. The clinical potential of miRNA lies in their fundamental tumoursuppressive and oncogenic functions and in their ability to modulate the efficacy of
cancer treatments, such as chemotherapy and radiotherapy. miRNAs have proven
to be efficient clinical diagnostic and prognostic markers in several human diseases
including cancer, as well as therapeutic targets of biological and chemical molecules. Studies on selective miRNA modulation through antisense inhibition,
mature miRNA replacement, chemical modification for better delivery or chemical
compounds could have a significant impact on the successful translation of
miRNA-based or miRNA-targeted therapeutics from bench to bedside. In recent
years, different experimental and computational approaches using methodologies
associated with miRNA detection and bioanalysis have therefore experienced rapid
growth. This section provides an overview of miRNA research methods and
technologies, mainly focusing on the latest and most advanced miRNA analysis
strategies.
Advanced Assays in Epigenetics
545
the DNA of interest linked to its interacting DNA. 4C technology is an advanced
3C-based approach, as it combines 3C with NGS technology. A further variant of 3C
is 5C technology, which has the added benefit of studying millions of interactions in
genomic areas by using universal primers in one single assay. 3C-based technologies
include Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIAPET), which combines the 3C method with ChIP and examines genomic sites
associated with specific transcription factors, which can be proximal and distal
[60]. In ChIA-PET, a specific antibody is used together with sonicated chromatin
and the protein of interest, and only this immunoprecipitated material linked to the
protein of interest is used to construct the ChIA-PET. Then, biotinylated oligonucleotides are used to ligate DNA proximal fragments, which are subsequently ligated
on beads. Finally, DNA is digested by a specific restriction enzyme which cuts to
release a tag-linker-tag region. After pull-down of biotinylated linkers by specific
beads, DNA tags are amplified. Hi-ChIP is an advanced Chia-PET method [61]
which provides a more accurate interpretation of information on genome conformation. In Hi-ChIP, DNA interactions are firstly established in situ in the nucleus before
lysis procedure. In this way it is possible to avoid false-positive interactions.
3 Epigenetic Regulatory Role of miRNA: New Analytical
Tools and Technologies
miRNAs are small non-coding RNAs whose role as actors and targets in epigenetic machinery is well established. They are efficient regulators of heterogeneous
physiological and pathological functions, as is well described in almost every
cancer model. The clinical potential of miRNA lies in their fundamental tumoursuppressive and oncogenic functions and in their ability to modulate the efficacy of
cancer treatments, such as chemotherapy and radiotherapy. miRNAs have proven
to be efficient clinical diagnostic and prognostic markers in several human diseases
including cancer, as well as therapeutic targets of biological and chemical molecules. Studies on selective miRNA modulation through antisense inhibition,
mature miRNA replacement, chemical modification for better delivery or chemical
compounds could have a significant impact on the successful translation of
miRNA-based or miRNA-targeted therapeutics from bench to bedside. In recent
years, different experimental and computational approaches using methodologies
associated with miRNA detection and bioanalysis have therefore experienced rapid
growth. This section provides an overview of miRNA research methods and
technologies, mainly focusing on the latest and most advanced miRNA analysis
strategies.
Advanced Assays in Epigenetics
545