filters include Varioskan (Thermo Scientific; Edmunds Optics filters: donor 450 nm
CWL, 25 mm diameter, 80 nm FWHM, Interference Filter and acceptor 1 in.
diameter, RG-610 Long Pass Filter) and EnVision (PerkinElmer; Chroma filters:
Emission Filter for EmSlot4).
2 Innovative Genome-Wide Technologies
Numerous accurate and reliable approaches have been gradually introduced to study
epigenetic processes both at the level of single genes and genome-wide, collectively
called epigenetic technologies [33]. Progress in epigenetics [34] is closely linked to
the design of new technologies. Since epigenetic alterations may cause several
disorders, studying the (epi)-genome is a valid approach to identify these deregulations. While the genome itself is stable, epi-modifications are in continuous transformation and so the choice of methodology is crucial. Genome-wide research
technologies [35] have dramatically enhanced the study of epigenetic phenomena
and the development of new epigenetic-based diagnostic and therapeutic tools.
Many innovative platforms associated with advanced software packages for the
study and interpretation of the epigenome are now available. These platforms are
often related to specific diagnostic panels for different diseases. This section will
describe the most advanced methodologies for epigenome-wide applications with a
particular focus on single-cell epigenomic methodologies [36].
2.1 ChIP: From Standard Procedures to Innovative
Applications
ChIP is one of the principal methodologies used to examine the epigenetic state of
chromatin, that is to say the regulatory processes including direct methylation of
DNA, covalent modifications to histones and protein interactions with genomic
regions. The standard ChIP protocol [37] includes a set of specific steps as shown
in Fig. 5. Over the years this technique has been extensively revised and improved to
provide a better “resolution” of epigenetic markers and profiles. However, ChIP
results can still be disappointing for several reasons: high background binding,
inappropriate controls, low affinity of antibodies, errors made during DNA amplification and suboptimal quality of reagents used in chromatin preparation procedures.
A further critical experimental point involves the starting material. Since
ChIP comprises numerous steps, the amount of cells is crucial and pooling
immunoprecipitated DNA samples is often a mandatory strategy. Sometimes pooling
starting material is not always possible as specific cell types, cancer cells or heterogeneous materials in general are used. The size of DNA fragments is another key
issue. DNA fragments are usually obtained by sonication and optimal fragment size
536
C. Dell’Aversana et al.
CWL, 25 mm diameter, 80 nm FWHM, Interference Filter and acceptor 1 in.
diameter, RG-610 Long Pass Filter) and EnVision (PerkinElmer; Chroma filters:
Emission Filter for EmSlot4).
2 Innovative Genome-Wide Technologies
Numerous accurate and reliable approaches have been gradually introduced to study
epigenetic processes both at the level of single genes and genome-wide, collectively
called epigenetic technologies [33]. Progress in epigenetics [34] is closely linked to
the design of new technologies. Since epigenetic alterations may cause several
disorders, studying the (epi)-genome is a valid approach to identify these deregulations. While the genome itself is stable, epi-modifications are in continuous transformation and so the choice of methodology is crucial. Genome-wide research
technologies [35] have dramatically enhanced the study of epigenetic phenomena
and the development of new epigenetic-based diagnostic and therapeutic tools.
Many innovative platforms associated with advanced software packages for the
study and interpretation of the epigenome are now available. These platforms are
often related to specific diagnostic panels for different diseases. This section will
describe the most advanced methodologies for epigenome-wide applications with a
particular focus on single-cell epigenomic methodologies [36].
2.1 ChIP: From Standard Procedures to Innovative
Applications
ChIP is one of the principal methodologies used to examine the epigenetic state of
chromatin, that is to say the regulatory processes including direct methylation of
DNA, covalent modifications to histones and protein interactions with genomic
regions. The standard ChIP protocol [37] includes a set of specific steps as shown
in Fig. 5. Over the years this technique has been extensively revised and improved to
provide a better “resolution” of epigenetic markers and profiles. However, ChIP
results can still be disappointing for several reasons: high background binding,
inappropriate controls, low affinity of antibodies, errors made during DNA amplification and suboptimal quality of reagents used in chromatin preparation procedures.
A further critical experimental point involves the starting material. Since
ChIP comprises numerous steps, the amount of cells is crucial and pooling
immunoprecipitated DNA samples is often a mandatory strategy. Sometimes pooling
starting material is not always possible as specific cell types, cancer cells or heterogeneous materials in general are used. The size of DNA fragments is another key
issue. DNA fragments are usually obtained by sonication and optimal fragment size
536
C. Dell’Aversana et al.