Table Of ContentDifferential Display
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Preface
When the technique of differential display was first described in the early 1990s,
sequence information existed for a few thousand human genes and some
functional information was available for most of those. At about that time, the
development of high throughput sequencing technologies and large genome data-
bases, including the development of EST (expressed sequence tag) databases and
the massive human genome project, greatly accelerated the process of accumu-
lation of genetic sequence data. While amounts of sequence information have
been growing logarithmically, however, information about the function of genes
has emerged much more slowly. Latest estimates suggest that there are as many
as 140,000 human genes in the genome, but to date we have some under-
standing of the functions of only about 5000 of these. In a very few years the
sequences of all human genes will be known, but there will be an information
bottleneck regarding the physiological activity of their protein products.
Differential gene expression technologies, like differential display, will play an
essential role in elucidating these functions.
Differential display was the first differential gene expression screening
technology to become accessible to a broad range of academic biologists. As a
novel technique, it suffered from some 'teething' problems; for example some
laboratories had difficulty reproducing their early results while others found a
high rate of false positives. As a result its reputation has suffered, perhaps
because in some cases it appeared to be too simple and straightforward so that
some early projects were embarked upon without enough preparation. Thus
some early experimental paradigms may not have been the most appropriate
ones to allow for correct interpretation of the data.
Despite early problems and disappointments, several thousand papers using
differential display have now been published. Many improvements in the
original technology have been described and more improvements are appearing
all the time. This book provides an overview of current differential display
technologies and suggests some new ways in which differential gene expression
will be studied in the future. Detailed protocols for techniques that have proved
successful in practice are supplied. The coverage includes recent advances in
fluorescent differential display, experimental design for analysis of complex
V
PREFACE
tissues, the RAP-array technique, efficient display of 3'-end cDNA fragments, and
cloning of differentially expressed genes.
Differential display, of course, has not developed in a vacuum and a number
of other techniques have been developed to achieve similar aims. For example,
techniques such as SAGE or DNA gridding ('microarray') technologies are expected
to replace differential display in due course. These technologies are relatively
immature however, and equipment is not yet generally available, especially to aca-
demic laboratories, for them. Nonetheless, it is important to be aware of these new
techniques and their capabilities, and this book descibes some of the new
developments in the area.
Ultimately the goal of molecular genetics as a discipline is to unravel the func-
tion of all the genes in the human genome (as well as other important genomes).
This book will allow researchers to make intelligent decisions about which differ-
ential gene expression technique to adopt for their particular project. Step-by-step
procedures are given to help ensure that projects use appropriate experimental
design, and that the best available techniques are followed in ways most likely to
result in success.
vi
Contents
List of protocols xiii
Abbreviations xvii
1 An introduction to differential display and related techniques 1
Harold A. Robertson and Ronald A. Leslie
References 4
2 Recent advances In fluorescent differential display 5
Karen Lowe
1. Introduction 5
Subtractive hybridization 5
Differential display 6
Random arbitrarily primed (RAP)-PCR 6
Representational difference analysis (RDA) 7
Subtractive display (SD) 7
2. Differential display strategy and primer design 8
HIEROGLYPH™ oligo(dT) anchored primers 10
HIEROGLYPH arbitrary primers 10
The importance of cDNA fragment length 1 \
3. Fluorescent differential display (fluoroDD) 12
Coverage with the fluoroDD system 15
A note on cDNA fragment expression patterns 15
Proper experimental design and RNA sample selection 16
Preparation of total RNA samples 16
First strand cDNA synthesis 20
Optimized fluorescent differential display with the fluoroDD system 22
4. fluoroDD gel electrophoresis and imaging 24
fluoroDD gel electrophoresis 24
fluoroDD gel analysis 26
Gel band selection and excision 27
5. cDNA fragment reamplification 28
6. Single strand conformation polymorphism (SSCP) gel 29
vii
CONTENTS
7. Direct cycle sequencing 32
8. Summary 32
Acknowledgements 32
References 33
3 Practical aspects of the experimental design for differential
display of transcripts obtained from complex tissues 35
Andrew D. Medhurst, David Chambers, Julie Gray, John B. Davis, Julian A Shale,
Ivor Mason, Peter Jenner, and Richard A. Newton
1. Introduction 35
2. Suitability of experimental system for differential display 37
Characterization of experimental model and definition of clear goals? 37
Determination of appropriate controls and sample replication 39
Selection of method for confirmation of differential expression 41
3. Preparation of samples for differential display 42
Tissue collection and homogenization 42
Total RNA extraction 43
DNase treatment 44
Reverse transcription 45
4. Generation of putative hits using differential display 46
Choice of equipment and reagents 46
Preliminary experiments 47
Differential display run 48
5. Processing of putative differentially displayed hits 51
Candidate band excision 51
Reamplification of candidate bands 53
Purification of reamplified products 55
TA cloning reamplified candidate bands 55
Colony selection and subclone identification 56
6. Confirmation of differential expression using independent techniques 58
References 64
4 RAP-array: expression profiling using arbitrarily primed probes
for cDNA arrays 67
Barbara Jung, Thomas Trenkle, Michael McClelland, Francoise Mathieu-Daude,
and John Welsh
1. Introduction 67
2. Generation of a RAP-PCR based reduced complexity probe to study
differential gene expression on cDNA arrays 70
RNA fingerprinting 70
Probing of RNA fingerprints on cDNA arrays 73
+
cDNA from poly(A) mRNA as probe on cDNA array 74
3. Arrays for studying differential gene expression 75
4. Hybridization to the array 76
viii
CONTENTS
5. Analysing arrays 79
6. Confirmation of differential gene expression using low stringency RT-PCR 79
References 81
5 The use of RT-PCR differential display In single-celled organisms and
plant tissues 83
Michael L Nuccio, Tzung-fu Hsieh, and Terry L Thomas
1. Introduction S3
2. Suitable total RNA preparation procedures 84
3. Differential display analysis 85
First strand cDNA synthesis 86
Differential display PCR amplification 87
Modifications to the PCR reaction 88
4. Band isolation, amplification, and downstream analysis 92
DNA cloning 94
Generation and use of differential display probes 97
5. Conclusion 98
References 99
6 A modified approach for the efficient display of 3' end restriction
fragments of cDNAs 101
Y. V. B. K. Subrahmanyam, Sigeru Yamaga, Peter E. Newburger, and Sherman M.
Weissman
1. Introduction 101
Gel display approaches to global analysis of gene expression 101
2. Methods 103
cDNA synthesis 103
Restriction enzyme digestion 108
Ligation of digested cDNA with Fly-Adapter 109
PCR amplification 111
Sequencing gel analysis of the PCR products 116
Recovery of differentially regulated cDNA bands from the dried gels for
sequencing 118
Processing of the PCR amplified samples for sequencing 123
Data documentation 125
3. Discussion 126
Acknowledgements 128
References 128
7 Cloning of differentially expressed brain cDNAs 131
Eileen M. Denovan-Wright, Krista L Gilby, Susan E. Howlett, and Harold A. Robertson
1. Introduction 131
2. Differential display protocols 132
References 147
ix
