T E C H N I C A L
T I P S
K. V. PHENIX, C. R. IRWIN, G. J. LINDEN, and J. J. MARLEY
School of Clinical Dentistry, The Queen‘s University of Belfast, Royal Victoria Hospital, Grosvenor Road,
Belfast BT12 6BP, Northern Ireland
Use of Expand™ High Fidelity for Differential Display
Introduction
With the introduction of recombinant
DNA techniques, there has been intense
interest in the identification of genes that
are expressed in particular cell types or
tissues under particular conditions and at
particular times. These “differentially
expressed” genes may be upregulated or
downregulated during cell cycling, cell
differentiation, or cell proliferation.
Differential gene expression also occurs
during cell/organ response to growth
factors, organic compounds, or such
pathophysiological conditions as carcinogenesis. The differentially expressed genes
may then be investigated to determine
their role in the process of interest.
The analysis of differentially expressed
genes, which previously required the
time-consuming method of subtraction
hybridization, can now be achieved with
differential display. The principle of
mRNA differential display is that, after
amplification of partial cDNA sequences
from subsets of mRNAs by RT-PCR, the
complexity of the cDNA population is
representative of the complexity of the
mRNA population from which it was
derived (1).
In the mRNA differential display
described by Liang and Pardee (1), the
mRNA content of two or more RNA
extracts is compared following reverse
transcription (RT) of the mRNAs with
oligo-(dT) primers anchored to the
beginning of the poly(A) tail and subsequent amplification in the presence of a
second 10-mer, arbitrary in sequence,
and a radiolabeled nucleotide. The
amplified cDNA subpopulations of the
3' termini of mRNAs as defined by the
specific pair of primers are then fractionated on a sequencing gel and visualized
by autoradiography. A comparison of the
resulting patterns enables the identification of PCR products that are differentially
represented in the various extracts.
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NO. 3 [1996]
CONTENTS
The resulting PCR products can then
be purified, re-amplified, and either
cloned or used as probes to screen cDNA
libraries or carry out in situ hybridization
studies. The use of numerous combinations of different primers in independent RT-PCR reactions ensures that
each mRNA present in the extract is converted to a defined PCR product.
Materials and Methods
In our laboratory, mRNA differential
display was performed essentially as
described by Liang and Pardee (1),
except for the choice of enzyme for PCR,
use of 33P (2), and design of primers. In
our example, we compared gene expression in early passage cultured fibroblasts
derived from gingival biopsy samples
taken from a patient with Cyclosporin Ainduced gingival overgrowth (GO6) to
cultured fibroblasts from an age/sex
matched normal control (NG3).
Total RNA was extracted from the cultured fibroblasts, and DNase I was used
to remove residual genomic DNA. For
each RNA sample, a reverse transcription
mixture was prepared with 1X reverse
transcription buffer, 10 mM dithiothreitol, 20 µM dNTPs, 0.2 µM H-T11C
primer (a one-base anchored primer with a
5 restriction site), 200 U M-MuLV
Reverse Transcriptase, and 1.5 U RNase
Inhibitor per 20 µl reaction volume. The
RNA was incubated at 65°C for 5 min,
chilled on ice, and 500 ng of DNase-free
RNA was added to the reverse transcription mix. Reverse transcription was carried out by incubation at 37°C for 60 min,
followed by 5 min at 95°C.
As mentioned above, one difference
from the Liang and Pardee method (1)
involved our design of primers. One-base
rather than two-base anchored oligo(dT) primers were used in an attempt to
reduce the redundancy and under-representation of certain mRNA species arising
l
as a result of the degeneracy of two-base
anchored oligo-(dT) primers (3). In addition, the inclusion of a restriction site
(Hind III) at the 5' end of the primers
gives rise to longer primers with the
ability to yield highly selective and reproducible displays of cDNA products that
are more readily manipulated for cloning
purposes.
To amplify duplicate cDNA samples,
polymerase chain reactions (PCR) were
performed on 0.1 volume of the reverse
transcription reactions with
Expand PCR buffer
1X
containing 1.5 mM MgCl2
dNTPs
2 µM
H-T11C primer
0.2 µM
appropriate arbitrary primer*
0.2 µM
Expand™ High Fidelity
1.5 U
Redivue™ [α-33P] dATP
(1000 – 3000 Ci/mmol)
1 µCi
dH20
to 20 µl
Total
20 µl
* For PCR, a series of 13-mers of arbitrary but
defined sequence were used in combination with
the H-T11C primer:
H-AP2: AAGCTTCGACTGT
H-AP3: AAGCTTTGGTCAG
H-AP4: AAGCTTCTCAACG
H-AP5: AAGCTTAGTAGGC.
The cDNAs were amplified under the
following cycling conditions:
Initial denaturation at 94°C for 5 min
■ 30 sec at 94°C
■ 2 min at 40°C
■ 30 sec at 72°C
for 40 cycles
Final extension for 5 min at 72°C.
PCR products were resolved on a 6%
polyacrylamide/8 M urea sequencing gel,
and the bands visualized by autoradiography.
31
T E C H N I C A L
T I P S
Results
By using Expand High Fidelity in
mRNA differential display, we were able
to compare differential gene expression
in normal and Cyclosporin-induced
overgrowth cultured gingival fibroblasts
(Figure 1).
References
1. Liang, P. and Pardee, A. B. (1992) Science 257:
967-971.
2. Phenix, K. V., Irwin, C. R., Linden, G. J., Marley, J. J.,
Read, C. A., and Somers, J. M. (1996) Life Science
News 19:11-12.
3. Liang, P., Zhu, W., Zang, X., Guo, Z., O’Connell,
R., Averboukh, L., Wang, F. and Pardee, A. B. (1994)
Nucleic Acids Research 22:5763-57.
Product
Cat. No.
Expand™ High
1 732 641
Fidelity PCR System*
1 732 650
Pack Size
100 U
(30 reactions)
2 x 250 U
(150 reactions)
10 x 250 U
(750 reactions)
1 759 078
Also Available
Deoxynucleotide
Triphosphate Set
Cat. No.
1 277 049
Pack Size
1 set (100 µl)
(4 x 10 µmol)
M-MuLV Reverse
Transcriptase
1 062 603
500 units
799 017
799 025
2000 units
10000 units
RNase Inhibitor
Figure 1 Differential display performed with Expand High Fidelity. Total RNA (500 ng) extracted from normal (N) and Cyclosporin-induced overgrowth (O) cultured gingival fibroblasts was reverse transcribed with a HT11C anchored primer. The resulting cDNAs were amplified by PCR, using the same H-T11C primer in combination
with four different arbitrary 13-mer primers (H-AP2, H-AP3, H-AP4, and H-AP5) as described in the text. The
amplified products were then fractionated on a 6% sequencing gel.
32
*This product is sold under licensing arrangements with
Roche Molecular Systems and The Perkin-Elmer
Corporation. Purchase of this product is accompanied by
a license to use it in the Polymerase Chain Reaction (PCR)
process in conjunction with an Authorized Thermal
Cycler.
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NO. 3 [1996]
CONTENTS