One of the most challenging tasks facing the modern biological research laboratory is to make sense of the enormous amount of data being generated by various genome projects currently underway, and especially the human genome project. Understanding the ways in which genes are differentially expressed in various tissues and cell types, throughout ontogenetic development and in pathological processes, will go a long way towards understanding the function of all these 'new' genes and their protein products. Differential Display explains in detail how to perform the technique of RT-PCR Differential Display in various kinds of experimental biological systems. It also examines this technique in the context of other methods of studying differential gene expression such as subtractive hybridisation and the use of high-density gene microarrays combined with hybridisation techniques and automatic image analysis.
List of protocols xiii Abbreviations xvii An introduction to differential display and related techniques 1(4) Harold A. Robertson Ronald A. Leslie References 4(1) Recent advances in fluorescent differential display 5(30) Karen Lowe Introduction 5(3) Subtractive hybridization 5(1) Differential display 6(1) Random arbitrarily primed (RAP)-PCR 6(1) Representational difference analysis (RDA) 7(1) Subtractive display (SD) 7(1) Differential display strategy and primer design 8(4) HIEROGLYPH™ oligo(dT) anchored primers 10(1) HIEROGLYPH arbitrary primers 10(1) The importance of cDNA fragment length 11(1) Fluorescent differential display (fluoroDD) 12(12) Coverage with the fluoroDD system 15(1) A note on cDNA fragment expression patterns 15(1) Proper experimental design and RNA sample selection 16(1) Preparation of total RNA samples 16(4) First strand cDNA synthesis 20(2) Optimized fluorescent differential display with the fluoroDD system 22(2) fluoroDD gel electrophoresis and imaging 24(4) fluoroDD gel electrophoresis 24(2) fluoroDD gel analysis 26(1) Gel band selection and excision 27(1) cDNA fragment reamplification 28(1) Single strand conformation polymorphism (SSCP) gel 29(3) Direct cycle sequencing 32(1) Summary 32(3) Acknowledgements 32(1) References 33(2) Practical aspects of the experimental design for differential display of transcripts obtained from complex tissues 35(32) Andrew D. Medhurst David Chambers Julie Gray John B. Davis Julian A. Shale Ivor Mason Peter Jenner Richard A. Newton Introduction 35(2) Suitability of experimental system for differential display 37(5) Characterization of experimental model and definition of clear goals? 37(2) Determination of appropriate controls and sample replication 39(2) Selection of method for confirmation of differential expression 41(1) Preparation of samples for differential display 42(4) Tissue collection and homogenization 42(1) Total RNA extraction 43(1) DNase treatment 44(1) Reverse transcription 45(1) Generation of putative hits using differential display 46(5) Choice of equipment and reagents 46(1) Preliminary experiments 47(1) Differential display run 48(3) Processing of putative differentially displayed hits 51(7) Candidate band excision 51(2) Reamplification of candidate bands 53(2) Purification of reamplified products 55(1) TA cloning reamplified candidate bands 55(1) Colony selection and subclone identification 56(2) Confirmation of differential expression using independent techniques 58(9) References 64(3) RAP-array: expression profiling using arbitrarily primed probes for cDNA arrays 67(16) Barbara Jung Thomas Trenkle Michael McClelland Francoise Mathieu-Daude John Welsh Introduction 67(3) Generation of a RAP-PCR based reduced complexity probe to study differential gene expression on cDNA arrays 70(5) RNA fingerprinting 70(3) Probing of RNA fingerprints on cDNA arrays 73(1) cDNA from poly(a)+ mRNA as probe on cDNA array 74(1) Arrays for studying differential gene expression 75(1) Hybridization to the array 76(3) Analysing arrays 79(1) Confirmation of differential gene expression using low stringency RT-PCR 79(4) References 81(2) The use of RT-PCR differential display in single-celled organisms and plant tissues 83(18) Michael L. Nuccio Tzung-fu Hsieh Terry L. Thomas Introduction 83(1) Suitable total RNA preparation procedures 84(1) Differential display analysis 85(7) First strand cDNA synthesis 86(1) Differential display PCR amplification 87(1) Modifications to the PCR reaction 88(4) Band isolation, amplification, and downstream analysis 92(6) DNA cloning 94(3) Generation and use of differential display probes 97(1) Conclusion 98(3) References 99(2) A modified approach for the efficient display of 3 end restriction fragments of cDNAs 101(30) Y.V.B.K. Subrahmanyam Sigeru Yamaga Peter E. Newburger Sherman M. Weissman Introduction 101(2) Gel display approaches to global analysis of gene expression 101(2) Methods 103(23) cDNA synthesis 103(5) Restriction enzyme digestion 108(1) Ligation of digested cDNA with Fly-Adapter 109(2) PCR amplification 111(5) Sequencing gel analysis of the PCR products 116(2) Recovery of differentially regulted cDNA bands from the dried gels for sequencing 118(5) Processing of the PCR amplified samples for sequencing 123(2) Data documentation 125(1) Discussion 126(5) Acknowledgements 128(1) References 128(3) Cloning of differentially expressed brain cDNAs 131(18) Eileen M. Denovan-Wright Krista L. Gilby Susan E. Howlett A. Robertson Introduction 131(1) Differential display protocols 132(17) References 147(2) Use of suppression subtractive hybridization strategy to identify differential gene expression in brain ischaemic tolerance 149(10) Xinkang Wang Giora Z. Feuerstein Introduction 149(1) Preparation of RNA for the SSH technique 150(1) The suppression subtractive hybridization technique 151(4) Summary 155(4) References 157(2) Studying gene expression profiles in specialized brain regions by microSAGE 159(22) Erno Vreugdenhil Nicole Datson Introduction 159(1) Steps in the SAGE procedure 160(21) References 180(1) Construction and applications of gene microarrays on nylon membranes 181(26) Jeffrey L. Mooney Paul S. Kayne Shawn P. OBrien Christine M. Debouck Introduction 181(2) Gene array construction on nylon membranes 183(8) Nylon membranes 183(1) Selection of test clones 183(1) Controls 184(2) DNA preparation 186(1) Robotics 187(1) Density 188(3) Probe preparation 191(5) RNA selection 191(1) RNA purification 191(3) Probe synthesis 194(2) Hybridization 196(2) Hybridization conditions 196(2) Image analysis and data interpretation 198(3) Data quantitation 198(1) Data interpretation 199(2) Construction and use of DNA microarrays on glass 201(3) Glass selection 202(1) DNA source 202(1) Microarray robotics 202(1) Microarray density 203(1) Probe preparation 203(1) Hybridization and detection 203(1) Data capture 204(1) Conclusion 204(3) References 205(2) Subtracted differential display and antisense approaches to analysing brain signalling 207(12) G.R. Uhl X.B. Wang Introduction: rationale for differential display approaches when a little, some, or a lot of the target genome is understood 207(5) Development of subtracted differential display (SDD) 208(4) Example: candidate drug-related genes from striatum 212(1) Antisense confirmation: one practical approach 212(2) Antisense design 212(1) Antisense administration 212(1) Confirmation of rGβ1 protein expression level 213(1) Evaluation of antisense effects on drug treated rats: effects on locomotor `sensitization 214(1) Pitfalls 214(1) Comparisons with other methods 215(1) Conclusions 216(3) References 217(2) Differential display analysis of memory-associated genes 219(30) A-Min Huang Eminy H.Y. Lee Introduction 219(1) Behavioural training and testing 220(1) Hippocampal tissue dissection and total RNA isolation 221(2) PCR differential display analysis 223(5) Reverse transcription 223(1) PCR amplification 224(1) Sequencing gel analysis 225(2) Recovery and reamplification of differentially expressed cDNA bands 227(1) Cloning and sequencing of the differentially expressed products 228(3) TA cloning 228(1) DNA sequencing 229(1) Database searching 229(2) Full-length cDNA cloning 231(1) Initial confirmation of PCR differentially expressed genes 231(5) Northern blot analysis 231(3) Reverse Northern blot analysis 234(1) Quantitative RT-PCR analysis 234(2) Further confirmation of PCR differentially expressed genes 236(9) In situ hybridization 237(3) Gene expression down-regulated by antisense oligonucleotide treatment 240(2) Gene knock-out study 242(3) Conclusions 245(4) Acknowledgements 246(1) References 246(3) List of suppliers 249(4) Index 253
