1. Microarray and DNA chips
for Transcriptome study
Amna Siddiqui,Areeba Khan, Faiq Sabih,
Hammad Naveed, Hassan Fajri
BBS-6

2. Overview
• Transcriptome study; why needed?
• Methods used
• What are Microarray and DNA chips?
• Difference between the two
• Method used to study one or more transcriptome
• Complications
• Alternate method to compare two transcriptomes
• Yeast transcriptome
• Applications

3. Transcriptome
A transcriptome is the collection of all
the mRNA transcripts transcribed
from the DNA (genome), at any one
time, in a particular cell.
• 4% of the total RNA content of the
cell
• Specifies the composition of the
proteome (concerned with coding
regions that will be expressed)
• Expression triggered by
environmental factors
• If conditions are not optimum, total
expression can be switched off.

4. Transcriptome study
• Study of the transcriptome of any organism’s cell is also
called expression profiling
• By analyzing transcriptome sequence, it can be
determined when and where a gene is turned off or on
in a particular cell
• Organisms may have same genes but different gene
expression leading to difference in behaviours
• Thus, comparison of two transcriptomes (from
different specie or different cell types) can be used to
determine properties of each cell type
• Doing transcriptome study of a diseased or cancerous
cell can lead to information about the gene responsible
for producing abnormality (gene expression less or
more)

5. Methods used in Transcriptome
Study
Serial Analysis of
Gene Expression
(SAGE)
Massively parallel
signature
sequencing (MPSS)
Microarray and
DNA chips
RNA-Seq
(emerging
method)

6. Microarray and DNA chips
• Microarray method used to check gene
expression
• Microarrays or DNA chips use a thin glass
microscopic slide, silicon chip or nylon membrane
• Thousands of reference genes can be
immobilized, spotted or synthesized in situ on a
small space on these glass slides
• Works on principle of hybridization of mRNA
(converted to cDNA) of concerned cell with
immobilized cDNA/oligonucleotide sequence
present on array slide

7. Microarray and DNA chips
• Advantage over SAGE: rapid evaluation of the
comparison of two transcriptomes can be
achieved by running them simultaneously on
identical arrays and checking hybridization
patterns of the two
• Further refined results can be achieved by using
mRNA that is bound to ribosome in cell.
Ribosome-bound mRNA gives part of
transcriptome that is actively being used in
protein synthesis

8. Difference between two
Microarray
• Uses PCR products or
cDNA of the genes of
interest
• Spotted on the surface of
the glass slide or nylon
membrane
DNA chip
• The oligonucleotides match
positions within the gene of
interest
• Mixture of oligonucleotides
synthesized in situ on the
surface of the glass slide or
silicon membrane.

9. Objectives of Microarray and
DNA chips
• Two major objectives
Objective:
Identify gene
whose
mRNA is
present
Microarray: PCR products or
cDNA derived from all genes of
interest
DNA chip: Mixture of
oligonucleotides synthesized
which match positions in the gene

10. Objectives of Microarray and
DNA chips
Objective:
Determine
relative
amounts of
mRNA
present
Each position in array contains up
to 109 copies of probe molecules.
Signal intensity at each position
will determine level of
hybridization of probe with
mRNA (high copy number, more
hybridization)

11. Basic Flowchart of Transcriptome
study using Microarray/DNA chip
Collect mRNA
molecules from a
cell
Use Reverse
Transcriptase (RT)
enzyme to produce
cDNA molecules from
the mRNA
Label cDNA with
flourescent dyes
Prepare
microarray/DNA chip
(cDNA from reference
genes or oligonucleotide
mixture)
Place labeled cDNA
on microarray slide
Hybridization of
labeled cDNA with
cDNA
(complimentary) on
microarray
Larger mRNA
amount in cell (more
expression), more
hybridization.Vice
versa
Scan array slide.
More flourescence,
more intensity of
expression

12. Microarray and DNA chip method

13. Microarray/DNA chip after hybridization. Color intensity
shows level of hybridization.The cDNA prepared from
mRNA is first labeled with fluorescent marker (like Cy3
and Cy5), then hybridized with array to produce such a
pattern.

14. Complications
• Hybridization analysis will have insufficient
specificity to distinguish between every mRNA
that could be present.
Two mRNAs, similar
sequences, may cross-
hybridize with each
other’s specific probe on
the array
Paralogous genes active
in same tissue – group of
related mRNAs can
hybridize with members
of the same gene family
Distinguishing which
specific mRNA is
present and how much
is present becomes
difficult
Two or more different
mRNAs could have been
derived from same gene
– alternate splicing
concept

15. Complications
• Alternate splicing

16. Complications
• When comparing more than one transcriptome,
differences in mRNA amount and hybridization
intensities must be due to difference in transcripts
rather than due to experimental errors.
• Experimental errors could include:
– Amount of target DNA on array
– Efficiency with which probe has been labeled
– Effectiveness of hybridization process
• Absolute precision and exact reproducibility of results
is almost impossible in different laboratories doing
same analysis (due to these experimental factors)

17. Normalization Procedures to
Counter Experimental Factors
• To counter these experimental factors, certain
normalization procedures are employed.
• Enables results from different array
experiments to be accurately compared
• Normalization procedure:
– Negative controls, so that background can be
determined in each experiment
– Positive controls, always give identical signals

18. Normalization Procedures to
Counter Experimental Factors
• In vertebrates, actin gene is used as positive
control
– Its expression level is fairly constant in any
particular tissue
– Even in developmental, or diseased state

19. Alternative method to study and
compare two transcriptomes
• Design experiment alternately
• Compare two transcriptomes directly, on single array
• Label the cDNA with different flourescent probes
• Scan the array at different wavelengths of light
• Determine relative intensities of the two types
fluorescence at each position
• Differences in the mRNA content of the two
transcriptomes can be directly analyzed.

20. Yeast transcriptome: An
example
• Ideally suited for transcriptome studies
• Little changes in yeast transcriptome, if
biochemical environment is constant
• Glucose rich – stable. Glucose depletion:
causes corresponding restructuring of
transcriptome.
• Transcriptome also undergoes restructuring
during cellular differentiation.
• Sporulation pathway.

21. Yeast transcriptome: An
example
• Spores adapt its mRNA at each stage to the
changing stressful conditions.
• Acts as model organism to study interactions
between genome and environmental signals in
higher eukaryotes.
• Transcriptome studies help to annotate a
genome sequence, helping in identifying gene
functions.

22. Applications
• Studying the transcriptome can lead to various
applications
– Transcriptomes of stem cell and cancer cells can
be studied by researchers to understand cellular
differentiation and carcinogenesis
– Transcriptomes of human oocytes and embryos
can be studied to understand molecular
mechanisms and signaling pathways in embryonic
development
– Used in biomarker discovery

23. Thank you!