Anita Rawat

Division of Genetics and Tree

Propagation
Forest Research Institute
Dehradun
 Hundreds of genes
(that contain information
for making proteins)
+
Regulatory sequences
(control how much of a gene will be made,
when it will be made,
and where in the body it will be made)
 Muscle Cell Pancreatic islet
Gene Gene Expression
cells
Expression Profile: Islet
Profile: Actin……..ON
Muscle Myosin……OFF
Actin……..ON Insulin……ON
Myosin……ON Melanin…..OFF
Insulin…… Cyclin 1…..ON
OFF Ubiquitin…ON
Melanin…..OF Histone 2B..ON
F
Cyclin 1…..ON
Nucleus Ubiquitin…ON Nucleus
DNA
Histone DNA
2B..ON

Gene 1 Gene 2 Gene3 Gene4 Gene 1 Gene 2 Gene3 Gene4

OFF ON ON OFF OFF ON OFF ON
Melanin Actin Myosin Melanin Actin Myosin
Insulin Insulin

mRNA mRNA

Actin Myosin Actin Insulin

DNA Microarray
 DNA microarrays are small pieces
of glass or silicon that have many
short pieces of DNA attached.

 Each short piece of DNA attached

to a microarray is called a probe .

 Every probe is different and

attaches specifically to one gene.

 Microarrays are often designed to

contain enough probes to detect
hundreds or thousands of
different genes.

 The precise location and

sequence of each probe is
recorded in a computer
database.

 Microarrays can be the size of a

microscope slide, or even
Three steps are
involved:
 Sequence the entire genome of the organism to be studied
 Design primer pairs for each gene
 Perform PCR to make copies of each gene
 Separate the double stranded DNA from each gene copy into
single strands
 Use robots to place microscopic droplets of each single
stranded DNA sample into ordered rows and columns on a
glass microscope slide. (This is the microarray)
 Create a database to keep a track of all the gene spots on the
microarray
 Make sure that all the spots contain equal amounts
of DNA.
 STEP 1. You first need to choose what cells or tissue
you want to study and grow them under specific
conditions. For example,
1. How gene expression changes when a plant is stressed for water.
2. What happens to the activity of various genes in yeast cells when
the cells are shifted from 25 ºC to 37 ºC?
3. What genes have increased expression in cancer cells compared to
normal cells?
 STEP 2. You isolate total mRNA from the cells you are
studying (both normal and treated or cancerous cells)
 STEP 3. Reverse transcribe the mRNA into cDNA and
introduce modified fluorescent bases into the DNA
(such as green and red)
 STEP 4. You mix the two populations of fluorescently
labeled cDNAs and hybridize them to the DNA chip and
then wash away all the unbound cDNA.
STEP 5. Using a scanner hooked to a computer
we measure how much of each labeled cDNA
(green and red) is bound to each spot on the slide.
The more label on a spot the more active the gene
General Scheme

IR64 IR64
well- stress
watered
panicles

extract

Total
RNA

cDNA
library
PCR 15 cycles

genes
mRNA from
Healthy plan

mRNA from
Stressed plan
 cDNA synthesis

Cyanine3 or Cy3 Cyanine5 or Cy5

(green) Fluorescent dye labeling (red)
Control/ well- Experimental/
RNase degrades mRNA
cDNA from healthy plant Sum total of cDNA cDNA from stressed plant
cDNA is applied to the chip and left overnight
for Hybridization
cDNA molecules hybridize to the complementary
sequences on the Chip
A grid is generated that uses a color code to
show the change in activity for each gene
By measuring the difference of intensity between
the two colors for each spot one can determine
whether a gene is more active or less active in a
treated cell compared to a normal cell.
 59 K oligo array from BGI, 10K rice panicle cDNA library printed at
Beijing IRRI

22K chips from Agilent

 Expresse
Induced d in both Repressed
conditions

Merged
images

R G
 GREEN represents Control DNA, where
either DNA or cDNA derived from normal
tissue is hybridized to the target DNA.
 RED represents Sample DNA, where
either DNA or cDNA is derived from
diseased tissue hybridized to the target
DNA.
 YELLOW represents a combination of
Control and Sample DNA, where both
hybridized equally to the target DNA.
 BLACK represents areas where neither
the Control nor Sample DNA hybridized to
the target DNA.
 R Test/ Experimental
∆ Gene expression
T
G Reference/
= Control

R 600 120 5600 1160 1300 1550 1800

0 0 0 0 0
G 1750 1650 1350 1090 650 250 800
0 0 0 0 0 0
0.0 0.0 0. 1. 2. 6. 22.5 0/0
3 7 4 0 0 2
What happens to the activity of various genes in
yeast cells when the cells are shifted from 25 ºC to
37 ºC?
hat genes have increased expression in cancer cells
compared to normal cells?

mRN
mRNA mRNA
mRNA
A

cDNA cDNA
 Can follow the activity of MANY genes at the
same time.
 Can get a lot of results fast
 Can COMPARE the activity of many genes in

diseased and healthy cells

 Can categorize diseases into subgroups
 Too much data all at once.
 Can take quite a while to analyze all the
results.
 The results may be too complex to
interpret
 The results are not always reproducible
 The results are not always quantitative
enough
 The technology is still too expensive
 Use of gene expression profiling in toxicology:
We can analyze the patterns of gene expression in tissues
exposed
to different chemicals.
 Use of gene expression profiling in ecotoxicology:
Gene expression profiles represent the primary level of
integration between environmental factors and the genome,
providing the basis
for protein synthesis, which ultimately guides the response of
organisms to external changes.
 cDNA microarrays can be used in heterologous
hybridizations:
The application of gene expression profiles is not limited to
model organisms for which the complete genome is available.
Several strategies are available to apply a genomic approach to
species for which only a limited amount of genomic information
is available.
 Applications of DNA microarrays in biology
Microarrays have been used to identify novel genes, binding
sites of transcription factors, changes in DNA copy number,
and variations from a baseline sequence, such as in
emerging strains of pathogens or complex mutations in
disease-causing human genes.
 Applications of DNA Microarray in Disease
Diagnostics
DNA microarrays have been used for genotyping and
determination of disease-relevant genes or agents causing
diseases, mutation analysis, screening of single nucleotide
polymorphisms (SNPs), detection of chromosome
abnormalities, and global determination of posttranslational
modification.
 The huge amount of data requires
standardization of storage, sharing, and
publishing techniques. To support the
public use and dissemination of gene
expression data, NCBI has launched the
Gene Expression Omnibus, or GEO. This
GEO is basically an expression data
repository and online resource for the
storage and retrieval of gene expression
data from any organism or artificial source.
 Recent Applications of DNA Microarray
Technology to Toxicology and
Ecotoxicology Environ Health Perspective.
2006 January; 114(1): 4–9.
 Applications of DNA microarrays in
biology. Annual Rev Biochemistry.
2005;74:53-82.
 NCBI. Microarray factsheet
 Applications of DNA Microarray in Disease
Diagnostics J. Microbiol. Biotechnol.
(2009), 19(7), 635–646
 Advantages and limitations of microarray
technology in human cancer Oncogene
(2003) 22, 6497–6507