The document discusses gene regulation, focusing on the transcriptome and the technologies used to analyze gene expression, particularly RNA sequencing (RNA-seq) and DNA microarrays. RNA-seq offers a comprehensive view of gene expression but is currently costly, while microarrays provide a snapshot of RNA levels and have been widely utilized since 1995. The document covers the principles, types, manufacturing processes, and applications of microarrays, alongside their advantages and disadvantages.

1. DNA
MICROARRAY

2. • transcription
• post transcription (RNA stability)
• post transcription (translational control)
• post translation (not considered gene regulation)
the “transcriptome”
Genes can be regulated at many levels
RNA PROTEIN
DNA
TRANSCRIPTION TRANSLATION
Usually, when we speak of gene regulation, we are referring to transcriptional
regulation. The complete set of all genes being transcribed are referred to as the
“transcriptome.”

3. In the last dozen years, it has become possible to look at
the entire transcriptome in a single experiment!
While there are a number of variations, there are
essentially two basic ways of doing this—using
sequencing-based methods and microarrays.
Sequencing-based methods are very powerful but have
typically been prohibitively expensive. However, with
recent advances in low-cost, high-throughput next
generation sequencing, these methods—referred to as
“RNA-seq”—are becoming more common and may soon
be dominant.

4. Genomic analysis of gene expression
• Methods capable of giving a “snapshot” of RNA
expression of all genes
• Can be used as diagnostic profile
– Example: cancer diagnosis
• Can show how RNA levels change during
development, after exposure to stimulus, during
cell cycle, etc.
• Provides large amounts of data
• Can help us start to understand how whole systems
function
Benfey and Protopapas, "Genomics" © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper
Saddle River, New Jersey 07458

5. Although details of the methods vary, the concept behind
RNA-seq is simple:
• isolate all mRNA
• convert to cDNA using reverse transcriptase
• sequence the cDNA
• map sequences to the genome
The more times a given sequence is detected, the more
abundantly transcribed it is. If enough sequences are
generated, a comprehensive and quantitative view of the
entire transcriptome of an organism or tissue can be
obtained.
RNA-seq

6. Microarrays may eventully be eclipsed by sequence-based
methods, but meanwhile have become incredibly popular since
their inception in 1995 (Schena et al. (1995) Science 270:467-70).
Microarrays are based on the ability of complementary strands of
DNA (or DNA and RNA) to hybridize to one another in solution with
high specificity.
There are now many variations. We’ll take a quick look at the two
basic types: Affymetrix (high density oligonucleotide) and glass
slide (cDNA, long oligo, etc). Both are conceptually similar, with
differences in manufacture and details of design and analysis.
DNA microarrays

7. Basics of microarrays
• DNA attached to solid
support
– Glass, plastic, or nylon
• RNA is labeled
– Usually indirectly
• Bound DNA is the
probe
– Labeled RNA is the
“target”
Benfey and Protopapas, "Genomics" © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper
Saddle River, New Jersey 07458

8. • A DNA microarray is composed of pieces of DNA ranging from 20-
5000 base pairs concentrated into specific areas on a solid support
such as a glass or silicon.
• Each spot of DNA, called a probe, represents a single gene.
• Each spot may contain a few million copies of identical probe
molecules.
• There are several synonyms of DNA microarrays such as DNA chips,
gene chips, DNA arrays, gene arrays and biochips.
• Microarray technology evolved from Southern blotting, where
fragmented DNA is attached to a substrate and then probed
• with a known DNA sequence.
• The use of microarrays for gene expression profiling was first
reported in 1995, and a complete eukaryotic genome
(Saccharomyces cerevisiae) on a microarray was published in 1997.

10. PRINCIPLE
• The principle of DNA microarrays lies on the
hybridization between the nucleotide.
• Using this technology the presence of genomic
or cDNA sequence in 100,000 or more sequences
can be screened in a single hybridization.
• The property of complementary nucleic acid
sequences is to specifically pair with each other
by forming hydrogen bonds between
complementary nucleotide base pairs.

12. Types of microarray
• DNA Microarray
• – cDNA microarray
• – Oligonucleotide arrays
• Protein microarray
• – Analytical
• – Functional
• – Reverse phase
• Chemical compound arrays
• – collection of organic chemical compounds spotted on a solid surface
• Carbohydrate arrays
• – various oligosaccharides and/or polysaccharides immobilized on a solid
• support in a spatially defined arrangement
• Cellular Microarrays
• – spotted with varying materials, such as antibodies, proteins, or lipids,
• which can interact with the cells, leading to their capture on specific
• spots

13. DNA microarray
• Thousands of small “spots” or “features,”
• Millions of strands of the same sequence within the spot
• covalently attached to the microarray surface
• The amount of DNA present in the spot correlates with the
• overall binding capacity of the spot
• The larger the binding capacity, the greater the amount of
• fluorescence signal that can be detected
• Binding capacity of the spot represents the detection range of the
microarray assay
• Each spot must contain sufficient binding sites to adequately
• represent differences in expression levels

14. • Spot is created by placing DNA “probes” on the
functionalized surface
• • Probes come in two distinct forms:
oligonucleotide and PCR probes (“cDNA probes”)
• • An oligonucleotide probe is a single-stranded
DNA that can range in size typically from 20 to 80
nucleotides in length and is synthesized using
standard phosphoramidite chemistry.
• • The cDNA probe is essentially a PCR product (of
almost any length) that is attached to the
microarray surface using a specific attachment
chemistry or simply ultraviolet cross-linking

15. • The decision to utilize oligo-probes or PCR probes depends upon
the amount of genomic information known about the organism
or cell system under investigation
• • It is nearly impossible to design oligo-probes for organisms
where no genomic data are available
• • Gene expression studies in “emerging” organisms (i.e., those
with little genomic data available) often involve PCR products
derived from a cDNA library
• • Advantages of oligo-probes are -multiple oligo-probes can be
designed to a single gene, targeting oligo-probe designs to
specific exons or exon boundaries to essentially avoid potential
cross hybridization with non-target genes

16. MANUFACTURING OF MICROARRAY SLIDES
• • Microarray analysis is invariably performed on
a glass slide, which enables to perform
hybridization assays with fluorescently labelled
samples.
• • Microarray manufacture requires three distinct
components:
• • 1. Production method
• • 2. Microarray slide
• • 3. Target genetic content

17. Microarray slides
• • Most commonly used support for microarrays are standard glass
microscope slides that offer flat and rigid support with low intrinsic
background fluorescence.
• • Nucleic acids will not attach efficiently to an untreated glass slide.
The treatment not only enable the binding of targets.
• • The uniformity and thickness of the surface coating on the slide is
• critical for good quality microarray results.
• • Variation in slide coating can contribute to the variation in
microarray signals and decrease the resolution of a microarray
experiment. Uneven slide coating can also lead to poor attachment
of deposited nucleic acid, which may come loose during microarray
hybridization.

18. Slide-based DNA microarrays
In general, slide-based arrays are used to make a direct comparison between two
different RNA samples. These can be a tissue sample vs. a reference, mutant vs. wild
type, treated vs. control, etc. The microarray provides a readout of the relative
differences in abundance of the RNAs present in each sample.
extract
mRNA
make
labeled
cDNA
hybridize to
microarray
cell type A
cell type B
more in “A”
more in “B”
equal in A & B

19. • Commonly used slide surface modifications include the introduction
of aldehyde, amino, or poly-lysine groups onto the slide surface.
• • Treated slides give highly consistent and reproducible data with
high signal to noise values, and they are most favorable for use in
• microarray experiments.
• Aldehyde slides
• • To minimize fluorescent background.
• • Aromatic amines on the G, C, and A bases of naturally occurring
DNA can also react with aldehyde groups.
• Amine slides
• • Amine groups can be introduced onto microarray slides by treating
• cleaned glass with aminosilane .
• • Vapor treatment of slides gives generally better results than
• deposition by a dipping method.

20. • Reflective slides
• • A large proportion of the fluorescent light
emitted from the hybridized probe is scattered in
all directions when using regular glass arrays.
• • The introduction of a reflective surface below
the spotting surface enables a significant amount
of this scattered output to be directed towards
the detector, hence increasing the amount of
signal detected by the system.
• • These reflective slides are constructed by adding
a layer of aluminium above the glass surface.

21. • Target nucleic acids
• • The third critical component in microarray manufacturing is the target
nucleic acid.
• • Microarray targets must be available in high enough concentration to
allow a sufficient number of molecules to be deposited onto the slide.
• • The purity of target solutions is important for both the efficient
attachment of nucleic acids to the slide surface and the availability of the
immobilized targets for hybridization.
• • PCR-amplified targets must be purified to remove dNTPs, primers, DNA
polymerase, buffer salts, and detergents.
• • The targets, once attached to the microarray surface, are only available
for hybridization when they are present in a denatured, single-stranded
form.
• • This can be achieved by spotting the targets under denaturing
• conditions, with in high salt solutions, or in denaturing solvents
• such as DMSO.

23. Sample Preparation for gene expression profiling by DNA
microarray
• • First step in sample preparation for gene expression
profiling is RNA isolation from the biological sample
• • The mRNA is converted to cDNA using reverse
transcription with fluorescently labelled nucleotides
• • These fluorescence-labeled cDNAs represent the mRNAs
in the original sample and are hybridized to the
microarray
• • The two fluorescent dyes typically utilized in
fluorescence labeling are cyanine-3 (Cy-3) and cyanine-5
(Cy-5), which are green- and red-colored dyes respectively

24. • Each microarray experiment involves two reverse transcription reactions
(e.g., control and drug-treated)
• • The “control” (e.g., untreated) mRNA sample is added to a reverse
transcription reaction that includes a dye-conjugated nucleotide (green)
• • Whereas the “test” (e.g., drug-treated) sample is added to a reverse
transcription reaction that includes a different dye-conjugated nucleotide
(red)
• • The cDNAs derived from the two reactions are mixed prior to microarray
hybridization, creating a “two-color” sample
• • if the test sample (i.e., drug-treated sample) causes GENE X to increase
the mRNA expression levels, then the GENE X spot will
• appear more red than green (after color channel normalization)
• • If the green fluorescence (control sample) from the GENE X spot is
measured at 10,000 relative fluorescence units (RFUs) and the red
fluorescence (test sample) at 40,000 RFUs, then the test sample contains a
fourfold increase in GENE X expression (i.e., a 400%
• increase over the control)

25. DNA microarray Hybridization
• • The hybridization method(s) are aimed at placing the fluorocDNA on
the two-dimensional surface utilizing a stringency conditions to facilitate
sequence specific binding
• • “Stringency” is a term used to describe the molecular
(thermodynamic) energy required for binding two complementary, single
stranded DNA molecules, which is dependent largely on temperature,
salt concentrations, and pH
• • High stringency conditions involve high temperatures and/or low salt
concentrations, and DNA hybridizations proceed slowly but in a
sequence specific manner
• • low stringency conditions involves cooler temperatures and/or
• high salt concentrations, and DNA can form double-stranded complexes
even if their sequences are not complementary (i.e., nonspecific binding)

26. • Hybridization involves placing the fluoro-cDNA in a specific buffer,
and sandwiching a sample volume (50–500 mL) between the DNA
microarray and a cover slip or blank glass slide
• • This assembly is then placed in a chamber where temperature,
and sometimes humidity, is controlled
• • Typically, the hybridization needs more than 16–19 h (i.e.,
overnight) to allow sufficient time for the probes to bind to the
fluoro-cDNAs in a sequence-specific manner
• • Once the incubation is complete, care should be taken while
removing the excess sample through a series of buffer washes
where stringency is controlled
• • Finally the microarrays (slides) are dried using centrifugation or
• airflow
• • The microarrays are now ready for scanning (i.e., fluorescence
• detection)

27. DNA Microarray Image processing
• • Microarrays are placed in a microarray scanning instrument
• • The spots will appear in varying colors from red to green to
yellow (yellow is a mixture of red and green fluorescence)
• • If the control sample was labeled green and the drug treated
• sample was labeled red, then a spot appearing red would
• indicate that the gene (mRNA)expression increased during
drug treatment
• • Spots lacking any color (fluorescence) indicate that the gene
(mRNA) was not expressed in the sample

28. • Once the microarray image has been derived
using the scanner (typically this is actually two
images representing the red and green images,
and the scanner software displays an “overlay”of
these images), raw data analysis is needed to
• – associate each spot with the gene (mRNA) that
it is detecting;
• and normalize the red and green channels to
correct for any differences in initial RNA
concentrations, labeling reaction efficiencies, and
differences in the capabilities of each channel (red
and green) within the scanner itself

29. • Preprocessing of oligo arrays generally involves three steps:
• background correction, normalization, and summarization
• • Normalization in microarray experiments is carried out based on
the assumption that only a small proportion of genes will be
differentially expressed among the thousands of genes present in the
array and/or that there is symmetry in the up- and down regulation
of genes
• • Most standard image processing algorithms extract the signal
intensities for each spot and from the surrounding background
• • The measurement of background intensities can be averaged over
entire arrays or taken from the area adjacent to a spot
• • The background intensity derived from the intensity values of the
lowest 2% of cells on the chip, establishes an overall
• baseline intensity to be subtracted from all cells before gene
• expression levels are calculated

30. Applications
• The DNA chips are used in many areas as given below:
• • Gene expression profiling (Transcriptome profiling)
• • Differential expression analysis
• • Diagnostics (Detection of SNPs, deletions and duplications) and
• genetic engineering
• • Analysis of post translational modifications (Alternative splicing
• detection)
• • Proteomics
• • Functional genomics
• • DNA sequencing
• • Toxicological research (Toxicogenomics)
• • Cellular profiling
• • Glycome analysis

31. ADVANTAGES
• Provides data for thousands of genes.
• • One experiment instead of many.
• • Fast and easy to obtain results.
• • Different parts of DNA can be used to study
gene expression.

32. Disadvantages:
• The biggest disadvantage of DNA chips is that they are
expensive to create.
• • The production of too many results at a time
requires long time for analysis, which is quite
complex in nature.
• • The DNA chips do not have very long shelf life,
which proves to be another major disadvantage of
the technology.
• •Identify gene expression of only those who already
reported.

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