68
C. Rochette-Egly
Development of the Field
During the last two decades, hybridization-based approaches (microarrays) in association with in silico approaches and high performance software analysis programs,
have been used to profile global gene expression patterns and RAR binding sites during RA-induced differentiation of ECCs and ESCs (Fig. 3.1). Such studies generated
a greater number of significant RA-target genes and novel RAREs, thus providing a
more comprehensive view of the regulatory events during cell differentiation.
Microarrays Analysis
Microarrays are a group of technologies in which specific DNA sequences or oligonucleotides are spotted or synthesized on a surface. Such arrays are used to probe a
mixture of labeled nucleic acids in order to measure the relative abundance of mRNA
transcripts (for review see Bumgarner 2013).
Originally, DNA microarrays (created in 1975) were produced using a colony
hybridization method. In the late 1990s and 2000s, the technology progressed rapidly
and it became possible to spot PCR-amplified cDNA clones on glass slides or nylon
membranes (DNA-Chip, Biochip) (Schena et al. 1995). Such arrays could be prepared by research scientists in their own labs to produce “in-house” custom-made
microarrays. These custom arrays could then be hybridized with cDNA prepared
from samples labeled with different fluorophores (for comparison purposes) and
scanned with equipment located in the researcher’s own laboratory (Fig. 3.2). Later,
oligonucleotide microarrays with high density gene representation (tens of thousands of genes to entire genomes) were generated (Lockhart et al. 1996) and commercialized (Affymetrix, GE Healthcare, Applied Biosystems, Agilent, Illumina and
others).
Using lab-generated, cDNA spotted arrays, Childs and colleagues characterized
hundreds of genes that are induced or repressed during RA-induced neural differentiation of P19 cells (Wei et al. 2002) and endodermal differentiation of F9
cells (Harris and Childs 2002). These genes were classified into functional groups
including transcriptional regulation, cell cycle control, extracellular matrix, signaling and metabolism (Table 3.1). Though some genes were RA-regulated in both cell
lines, numerous ones were found to be involved in lineage specification (neurons or
endoderm). In addition, the identified genes depicted distinct patterns of temporal
expression suggesting the existence of different phases in ECCs differentiation.
Commercialized, cDNA-based arrays with hundreds of cDNAs spotted on nylon
membranes (Atlas
® and UniGene cDNA arrays) also revealed hundreds of RAregulated genes during the neuronal differentiation of mouse (m) ESCs (Kelly and
Rizzino 2000) and human (h) NT2/D1 ECCs (Freemantle et al. 2002; Table 3.1).
When a Clontech Atlas
® Mouse 1.2 cDNA array and an oligonucleotide
Affymetrix Murine Genome U74Av2 GeneChip
® (with ten times more genes i.e.12
C. Rochette-Egly
Development of the Field
During the last two decades, hybridization-based approaches (microarrays) in association with in silico approaches and high performance software analysis programs,
have been used to profile global gene expression patterns and RAR binding sites during RA-induced differentiation of ECCs and ESCs (Fig. 3.1). Such studies generated
a greater number of significant RA-target genes and novel RAREs, thus providing a
more comprehensive view of the regulatory events during cell differentiation.
Microarrays Analysis
Microarrays are a group of technologies in which specific DNA sequences or oligonucleotides are spotted or synthesized on a surface. Such arrays are used to probe a
mixture of labeled nucleic acids in order to measure the relative abundance of mRNA
transcripts (for review see Bumgarner 2013).
Originally, DNA microarrays (created in 1975) were produced using a colony
hybridization method. In the late 1990s and 2000s, the technology progressed rapidly
and it became possible to spot PCR-amplified cDNA clones on glass slides or nylon
membranes (DNA-Chip, Biochip) (Schena et al. 1995). Such arrays could be prepared by research scientists in their own labs to produce “in-house” custom-made
microarrays. These custom arrays could then be hybridized with cDNA prepared
from samples labeled with different fluorophores (for comparison purposes) and
scanned with equipment located in the researcher’s own laboratory (Fig. 3.2). Later,
oligonucleotide microarrays with high density gene representation (tens of thousands of genes to entire genomes) were generated (Lockhart et al. 1996) and commercialized (Affymetrix, GE Healthcare, Applied Biosystems, Agilent, Illumina and
others).
Using lab-generated, cDNA spotted arrays, Childs and colleagues characterized
hundreds of genes that are induced or repressed during RA-induced neural differentiation of P19 cells (Wei et al. 2002) and endodermal differentiation of F9
cells (Harris and Childs 2002). These genes were classified into functional groups
including transcriptional regulation, cell cycle control, extracellular matrix, signaling and metabolism (Table 3.1). Though some genes were RA-regulated in both cell
lines, numerous ones were found to be involved in lineage specification (neurons or
endoderm). In addition, the identified genes depicted distinct patterns of temporal
expression suggesting the existence of different phases in ECCs differentiation.
Commercialized, cDNA-based arrays with hundreds of cDNAs spotted on nylon
membranes (Atlas
® and UniGene cDNA arrays) also revealed hundreds of RAregulated genes during the neuronal differentiation of mouse (m) ESCs (Kelly and
Rizzino 2000) and human (h) NT2/D1 ECCs (Freemantle et al. 2002; Table 3.1).
When a Clontech Atlas
® Mouse 1.2 cDNA array and an oligonucleotide
Affymetrix Murine Genome U74Av2 GeneChip
® (with ten times more genes i.e.12