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15 views

Microarray PDF

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2808ayush
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Anita Rawat

Division of Genetics and Tree Propagation


Forest Research Institute
Dehradun
DNA is located in a compartment of the cell called the
nucleus and is packaged in structures called
chromosomes

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)
Although all cells in the human body contain the same
DNA, but the cell types in the body vary widely.

Differences among cells are not due to the differences in


the DNA, but rather due to the differences in the amount
and types of genes that the cell expresses.
Muscle Cell Pancreatic islet cells
Gene Expression Gene Expression
Profile: Muscle Profile: Islet
Actin……..ON Actin……..ON
Myosin……ON Myosin……OFF
Insulin……OFF Insulin……ON
Melanin…..OFF Melanin…..OFF
Cyclin 1…..ON Cyclin 1…..ON
Ubiquitin…ON Ubiquitin…ON
Histone 2B..ON Histone 2B..ON

Nucleus Nucleus
DNA DNA

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


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

mRNA mRNA

Actin Myosin Actin Insulin


DNA Microarray
 DNA microarray analysis is a technique that
scientists use to determine whether genes are on
or off.

 The human genome contains approximately 40,000


genes. At any given moment, each of our cells has
some combination of these genes turned on, and
others are turned off.

 We can figure out which are on and which are off


by gene expression profiling, using a technique
called DNA MICROARRAY ANALYSIS.

 Microarray analysis involves breaking open a cell,


isolating its genetic contents, identifying all the
genes that are turned on in that particular cell
and generating a list of those genes.
 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 smaller.
I. Preparing a DNA
chip

Three steps are involved: II. Carrying out the


reaction (isolate
mRNA, process the
mRNA and hybridize
to DNA chip)

III. Collecting and


analyzing the
results
 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 well- IR64


watered stress
panicles

extract

Total RNA

cDNA library

PCR 15 cycles

genes
mRNA from
Healthy plant

mRNA from
Stressed plan
cDNA synthesis

Cyanine3 or Cy3 (green) Cyanine5 or Cy5 (red)


Control/ well-watered Fluorescent dye labeling Experimental/ stressed
plant plant
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, Beijing 10K rice panicle cDNA library printed at IRRI

22K chips from Agilent


Expressed
Induced 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 1200 5600 11600 13000 15500 18000

G 17500 16500 13500 10900 6500 2500 800

0.03 0.07 0.4 1.0 2.0 6.2 22.5 0/0


What happens to the activity of various genes in yeast
cells when the cells are shifted from 25 ºC to 37 ºC?
What genes have increased expression in cancer cells
compared to normal cells?

mRNA
mRNA mRNA
mRNA

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.
Renn et al (2004) have used heterologous hybridization to study gene
expression profiling across a wide range of different species of African
cichlid fish.
 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

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