Differential display

polymerase chain reaction

Course directors: Prof.Dr. Hala Eissa & Dr. Heba Radwan.

Prepared by: Dr. Nermin
Demonstrators: Eman, Nesma, Heba, Mawada, Dina
• Since its invention 1992 by Liang and Pardee,
differential display (DD) has become one of the
most commonly used techniques for identifying
differentially expressed genes at the mRNA level.
• DD systematically detects changes in mRNA profiles
among multiple samples being compared without
the need of any prior knowledge of genomic
information of the living organism being studied.
• The mRNA differential display technique has become a popular method
for isolating novel genes in a variety of biological systems and
comparing gene expression in different tissues or differently treated
samples.
• Yield results in only 1 - 3 days.

May be novel genes

• The mRNA differential display technique permits
simultaneous identification of both up. and
down-regulated genes.
Experimental design
• The experiment must be designed by establishing
a set of conditions which will be used as a
benchmark for the study.
Experimental design
● Control ● Condition 1 ● Condition 2
● Replicate 1 ● 3 weeks wheat 3 weeks wheat 3 weeks wheat
plant grown plant grown plant grown
● Replicate 2 under 16 hours under 16 hours of under 16 hours of
● Replicate 3 of illumination illumination and illumination and
exposed to exposed to
salinity stress drought stress
●
Steps of DD
• After applying the treatment, the steps will be as
follows:
1. Total RNA isolation
2. Reverse transcription of mRNA
3. PCR amplification
4. Polyacrylamide gel electrophoresis
5. Isolation of PCR fragment
6. PCR reamplification
7. Cloning
8. Sequencing
9. Analysis
Detailed steps
1. Total RNA isolation

Trizol method

Lithium chloride method

Spin column method

RNA extraction critical considerations

• Always wear clean gloves (don’t touch your

face/hair or “dirty” surfaces with your gloves).
• Use filter tips and other RNase-free consumables.

• Make sure the pipette barrel is also nuclease-free.

• Use DEPC-treated water at all times.

(note that DEPC is highly toxic)
• DNase treatment must be done
2-RT-PCR

• By using oligo (dt) anchor primer

• GTTTTTTTTT ATTTTTTTTT CTTTTTTTT

• Reverse transcriptase to
synthesis first strand cDNA
Detailed steps
2. Reverse transcription of mRNA
mRNA

5’ UTR Coding region 3’ UTR AAAAAAAn

UCG

Reverse transcription
MMLV

Note: for each RNA set up four reactions (one tube for each degenerate
anchored oligo(dT) primer set - T12MA,T12MC, T12MG, T12MT; where M
is A,C or G)
Detailed steps
2. Reverse transcription of mRNA
mRNA
Anchor Primer

5’ UTR Coding region 3’ UTR AAAAAAAn

UCG

Reverse transcription
Detailed steps
2. Reverse transcription of mRNA
mRNA
Anchor Primer

5’ UTR Coding region 3’ UTR AAAAAAAn

UCG

Reverse transcription
Detailed steps
2. Reverse transcription of mRNA
mRNA
Anchor Primer

5’ UTR Coding region 3’ UTR AAAAAAAn

UCG

Reverse transcription

cDNA(s)
5’ UTR Coding region 3’ UTR AAAAAAAn
UCG
 TTTTT
RT reaction – step by step
TTTTT
AAAAA RNase
TTTTT inhibitor
TTTTT
dATP
dCTP
ºC 65 dGTP
denature .1 dTTP
AAAAA
mRNA
RT Reverse
transcriptase

anneal + .2 ºC 37
elongate
AAAAA
mRNA

cDNA TTTTT

ºC 37

AAAAA
mRNA TTTTT
RT RT
cDNA
ready
Detailed steps
3. PCR amplification
Anchor Primer
cDNA(s)

5’ UTR Coding region 3’ UTR AAAAAAAn

UCG

Arbitrary Primers
(80)
PCR Amplify

labeled dNTPs

X Y Z
Detailed steps
3. PCR amplification

Setup a series of trial PCRs to establish the optimum

concentrations of control and test RNAs required to produce
a pattern of 100-300 amplified cDNA bands after gel
electrophoresis and autoradiography.
•
 RT–PCR at the bench
:RT
:Add
65ºC – 10 min ºC – 1 hour 37
RNasin
total RNA +
oligodT
RT
dNTPs
denature
Enzyme
anneal +
elongate
ready

:PCR ul 1-5

template 95ºC – 30 sec

95ºC 55ºC – 30 sec 72ºC
Buffer min 3 72ºC – 1 min min 10
MgCl2
PCR
cycles 30
dNTPs ready
denature amplify finish
DNA pol

primers

Gel analysis
Detailed steps
4. Polyacrylamide gel electrophoresis

Autoradiography image of
differential display gel

Silver staining ??
Detailed steps
4. Polyacrylamide gel electrophoresis
•
Polyacrylamide gel must be treated with urea
(denaturation) and DNA is treated with formamide.
•
Detailed steps
4. Polyacrylamide gel electrophoresis
• Detection of cDNA band on sequencing gel:

▫ Radioactive with autoradiography

detection.

▫ Fluorescence labelling techniques.

•
 Microtrough System

• Horizontal electrophoresis
system with 132 lanes.
• Containing grooved glass plates.
• Allows one to load at least one Sequencing gel unit
entire 96-well plate on one gel.
• Hand position during loading is
more stable and relaxed with
this system.

• Microtrough
•
Detailed steps
5. Isolation of PCR fragment
5. isolation of PCR fragment
Advantages of differential display
•

(1)Simplicity: Technically, it is based on PCR and DNA

sequencing gel electrophoresis.

(2) Sensitivity: Five to ten micrograms of total RNA is

enough to cover the majority of mRNAs in a eukaryotic

cell.

(3) Reproducibility: Up to 99% of the bands on a mRNA

display are reproducible.

Advantages of differential display
(4) Versatility: More than two RNA samples can be compared

simultaneously allowing complex comparisons in the same experiments.

(5) Detecting Novel Genes: Unlike microarray, DD does not require prior

knowledge of the mRNA sequence to be detected. Therefore, DD is

considered an "open system" versus the "closed system" of Microarrays.

(6) Speed and Expense: Within two days, the pattern of mRNA display

can be obtained.