GENE

EXPRESSION
STUDIES
 INTRODUCTION
• Gene expression is the process by which
information from a gene is used in the
synthesis of a functional gene product that
enables it to produce end products.

 The study of gene regulation provides

insights into normal cellular processes, such
as differentiation, and abnormal or
pathological processes.
 • Gene expression analysis studies
GENE can be studied by:
EXPRESSION 1. Northern blotting
PROFILING AND 2.DNA microarray
QUANTITATION:
METHODS AND 3. PCR, RT PCR
TECHNIQUES 4. NGS
 RNA EXPRESSION
• Northern blotting : The Northern blot is a technique used
in molecular biology research to study gene expression in a
sample, through detection of RNA (or isolated messenger
RNA )

• Principle : The fundamental principle of northern blotting

is to separate RNA based on their size using gel
electrophoresis and identified on a cellular membrane by
means of hybridization probe with a base sequence
corresponding to all or a part of the chain of the target RNA
STAGES OF
NORTHERN
BLOTTING
 ADVANTAGES OF NORTHERN BLOTTING
• Northern blots are particularly useful to determine conditions under
which specific genes are expressed.
• It is also useful in detection of mRNA transcript size.
• Blots can be stored for several years and reprobed if necessary.
 DISADVANTAGES OF NORTHERN BLOTTING
• Risk of mRNA degradation during electrophoresis: quality and
quantification of expression are negatively affected.
• High doses of radioactivity and formaldehyde are a risk for workers
and the environment.
• The sensitivity of northern blotting is relatively low in comparison with
that of RT-PCR.
• Detection with multiple probes is difficult and also time consuming
procedure .
• Use of ethidium bromide, DEPC and UV light needs special training
and attention.
 DNA MICROARRAYS
• A microarray is a laboratory tool used to detect the expression
of thousands of genes at the same time. DNA microarrays are
microscope slides that are printed with thousands of tiny spots
in defined positions, with each spot containing a
known DNA sequence or gene.
• Principle :
The core principle behind microarrays is hybridization between
two DNA strands, the property of complementary nucleic acid
sequences to specifically pair with each other by
forming hydrogen bonds between complementary nucleotide
base pairs. After washing off non-specific bonding sequences,
only strongly paired strands will remain hybridized.
Fluorescently labeled target sequences that bind to a probe
sequence generate a signal that depends on the hybridization
conditions (such as temperature), and washing after
hybridization
 PRINCIPLES OF DNA MICROARRAY TECHNOLOGY
• The principle of DNA microarray technology is based on the fact
that complementary sequences of DNA can be used to hybridise,
immobilised DNA molecules.
• There are four major steps in performing a typical microarray
experiment.
1.Sample preparation and labeling
2. Hybridisation
3.Washing
4. Image acquisition and Data analysis
 EFFECT OF STIMULI TO THE NORMAL CELL

Stimuli (Environment/
Drug/Stress) What will happen to the
cell ?

 Cell’s physiology?
Normal cell
 Cell’s biochemical?
 Molecular mechanisms?
 Expression profiling?

Cancerous
cell
HOW WOULD YOU MEASURE GENE EXPRESSION
PROFILING OF EXPERIMENTAL SAMPLE?
Tools of FG:
Gene expression profiling!!
Cancerous DNA-microarray
cell Responsible genes: technology
Up-regulated genes Real-time PCR
Down-regulated genes NGS

Normal Cell

Comparison between Cancer cell’s expression profiling with Normal cell’s

expression - ?? Up-regulated genes; ?? Down-regulated genes
1) DNA-MICROARRAY TECHNOLOGY
DNA-chip or biochip.
Is a collection of microscopic DNA
spots attached to a solid surface.
 To measure the expression levels of
large numbers of genes
simultaneously.
TYPES OF DNA MICROARRAYS
Two types of arrays exist:
1. DNA-macroarray: A collection of cDNAs on nylon membrane.
 Easily available, less expensive, reusable
Limitation:
 1000 spots
 Use of radioactive isotopes
 High background
2. DNA-Microarray : A collection of cDNAs on glass or silicon biochip.
>100,000 genes spots, no background signal
Limitation:
Costly
Generate huge data
Can’t be reusable
STEPS TO BE PERFORMED !
Experimental setup Factors affecting expression profiling

Sample

Tissue /Cells Amount of sample

Sample preparation Methods/Kits

Method of qualitation and
RNA Isolation quantitation
DNA microarray Chip RNA Qualitation and Quantitation Choice of reverse transcriptase
cDNA/Oligonucleotides Reverse Transcription
cDNA Labelling

Time Length
Hybridization and scanning Temperature
Component of Washing
Blocking , Stringency Washing buffer
PMT gain settings

Result Analysis

Normalization, Statistical Analysis

 EXPERIMENTAL DESIGNING
 equires reference sample
 Biological repeat = 03 (to remove
biasness)
 Sample may be pooled
 Technical repetition is further
needed A: day light 8 hrs B: day light 2 hrs

NORMAL EXPERIMENTAL

Pool Pool

1 2 3 1 2 3
SAMPLE PREPARATION (TOTAL RNA
ISOLATION)
Two known methods:
1.GITC: Phenol:Chloroform
precipitation method
2.Silica
Gel bead based
method
 Quantification

 Quality assessment
 Purity assessment
Muyal et al. BMC Diagnostic Pathology 2009; 4: 9
 TOTAL RNA PURITY AND QUANTITATION
RNA Quality = on the basis of
28S and 18S rRNA bands
RNA Quantity = OD at 260nm
RNA Purity =
OD260nm/OD280nm = >1:8 and
<2:1

For successive hybridization, 20µg

total RNA is required.

RNA limitation problem!

RNA Pre-amplification technique
Amplify 1000x of the initial
CDNA SYNTHESIS AND LABELING WITH
CY3/CY5 DYES

Control Test sample

Pool Pool

1 2 3 1 2 3

Cy3 Cy5

Direct method
HYBRIDIZATION AND SCANNING
 ANALYSIS: GENEPIX PRO Cy5 = 100,000
Cy3 = 20,000
Cy5/Cy3 =
5.0x

Cy5 = 20,000
Cy3 = 100,000
Cy5/Cy3 =
0.2x
Range = >2.0 (up-regulation; <0.5 (down-regulation)
• Reverse Transcription PCR : steady-state levels of mRNA are
quantitated by reverse transcription of the RNA to cDNA followed by
quantitative PCR (qPCR) on the cDNA. The amount of each specific
target is determined by measuring the increase in fluorescence
signal from DNA-binding dyes or probes during successive rounds of
enzyme-mediated amplification.

• Next-generation sequencing (NGS) is a massively parallel

sequencing technology that offers ultra-high throughput, scalability,
and speed. The technology is used to determine the order of
nucleotides in entire genomes or targeted regions of DNA or RNA.
 PROMOTER ANALYSIS

• Expression of reporter genes/promoter fusions in host

cells — promoter activity (transcription rate) is measured in
vivo by introducing fusions of various promoter sequences with
a gene encoding a product that can be readily measured to
monitor activity levels
• In vitro transcription (nuclear run-on assays) —
transcription rates are measured by incubating isolated cell
nuclei with labeled nucleotides, hybridizing the resultant
product to a membrane (slot blot), and then exposing this to
film or other imaging media
• Gel shift assays — also called electrophoretic mobility
shift assays, these are used to study protein-DNA or
protein-RNA interactions. DNA or RNA fragments that are
tightly associated with proteins (such as transcription
factors) migrate more slowly in an agarose or
polyacrylamide gel (showing a positional shift). Identifying
the associated sequences provides insight into gene
regulation
 PROTEIN EXPRESION
• Western blotting — quantification of relative expression levels for
specific proteins is accomplished by electrophoretically separating
extracted cell proteins, transferring them to a membrane, and then
probing the bound proteins with antibodies (targeted to antigens of
interest) that are subsequently detected using various chemistries or
radiolabelling
• 2-D Gel Electrophoresis — protein expression profiling is achieved by
separating a complex mixture of proteins in two dimensions and then
staining to detect differences at the whole-proteome level
• Immunoassays — proteins are quantitated in solution using antibodies
that are bound to color-coded beads (as in the Bio-Plex supension array
system) or immobilized to a surface (ELISA), which is subsequently
probed with an antibody suspension and is typically detected using a
chromogenic or fluorogenic reporter
 POSTTRANSLATIONAL MODIFICATION
ANALYSIS
• Immunoassays — levels of protein phosphorylation and
other post-translational modifications are detected using
antibodies that are specific for these adducts
• Mass spectrometry — proteins and their modifications
are identified based on their mass.
 DNA MICROARRAYS
• A microarray is a laboratory tool used to detect the
expression of thousands of genes at the same time. DNA
microarrays are microscope slides that are printed with
thousands of tiny spots in defined positions, with each spot
containing a known DNA sequence or gene.
• Principle :
The core principle behind microarrays is hybridization between
two DNA strands, the property of complementary nucleic
acid sequences to specifically pair with each other by
forming hydrogen bonds between complementary nucleotide
base pairs. After washing off non-specific bonding
sequences, only strongly paired strands will remain
hybridized. Fluorescently labeled target sequences that bind
to a probe sequence generate a signal that depends on the
hybridization conditions (such as temperature), and washing
after hybridization
 PRINCIPLES OF DNA MICROARRAY TECHNOLOGY
• The principle of DNA microarray technology is based on the fact
that complementary sequences of DNA can be used to hybridise,
immobilised DNA molecules.
• There are four major steps in performing a typical microarray
experiment.
1.Sample preparation and labeling
2. Hybridisation
3.Washing
4. Image acquisition and Data analysis
 • Drug discovery
APPLICATIO • Study of functional genomics
NS OF DNA • DNA sequencing
MICROARRA • Gene expression profiling
Y
• Study of proteomics
TECHNIQUE:
DRUG • Diagnostics and genetic engineering
DISCOVERY • Toxicological researches
• Pharmacogenomics
 REVERSE TRANSCRIPTASE PCR

• RT-PCR, also known as Reverse Transcriptase

PCR, is a variation of the polymerase chain
reaction that typically measures RNA
expression levels. In RT-PCR, complementary
DNA (cDNA) is made by reverse
transcribing of the RNA templates with the
enzyme reverse transciptase.
• This technique is used to qualitatively study
gene expression, and can be combined with
real time PCR (qPCR) to quantify RNA levels.
 PRINCIPLE
• Reverse transcription and PCR amplification can be performed as a two-step process in
a single tube or with two separate reactions. In both cases, RNA is first reverse-
transcribed into cDNA, which is then used as the template for PCR amplification.
• The primers used for cDNA synthesis can be either non–sequence-specific primers (a mixture
of random hexamers or oligo-dT primers) or sequence-specific primers.
• Non-sequence-specific primers:
• Random hexamers are a mixture of all possible combinations of six nucleotide sequences
that can attach randomly to mRNA and initiate reverse transcription of the entire RNA pool.
• Oligo-dT primers are complementary to the poly-A tail of mRNA molecules and allow
synthesis of cDNA only from mRNA molecules.
• Sequence-specific primers:
• Sequence-specific primers are the most restricted because they are designed to bind
selectively to mRNA molecules of interest, which makes reverse transcription a target-specific
process.
ONE STEP
V/S TWO
STEP
RT-PCR
 APPLICATIONS OF RT-PCR

Many clinically important viruses have genomes composed of RNA, RT-PCR is useful
for detecting such viruses. RT-PCR has also been used for the detection of the viral
causes of meningitis and meningoencephalitis, such as enteroviruses and the West
Nile virus. RT-PCR is being used for the detection of the following viruses:
•Dengue virus
•Hantavirus

•Human metapneumovirus
•Severe acute respiratory syndrome (SARS)
•QuantitativeRT-PCR assays are commonly used for the detection of HIV and
HCV viral load (amount of these viruses present in the blood of a patient) testing.