detecting differences in gene expression
TRANSCRIPT
and to students approaching endocrinology as ascientific discipline. For those who require a moreclinically intensive text, Basic and ClinicalEndocrinology1 is a concise, single-volume textthat, in the manner of this book, also fills the gapbetween simple primer and bulky, encyclopaedictexts such as Endocrinology2 and Williams’Textbook of Endocrinology3.
References01 Greenspan, F.S., and Strewler, G.J., eds (1997)
Basic and Clinical Endocrinology, 5th Edition,Prentice-Hall International.
02 DeGroot, L. et al., eds (1995) Endocrinology (3rdedn), W.B. Saunders
03 Wilson, J.D. and Foster, D.W., eds (1992) Williams’Textbook of Endocrinology (8th edn), W.B. Saunders
Rory J. Clifton-Bligh BSc(Med), MB, BSEndocrine Fellow
Dept of Medicine, Level 5, Addenbrooke’sHospital, Cambridge, UK CB2 2QQ.
Tel: +44 1223 336 853Fax: +44 1223 336 846
e-mail: [email protected]
13
ResourcesMOLECULAR MEDICINE TODAY, JANUARY 1998
Copyright ©1998 Elsevier Science Ltd. All rights reserved. 1357 - 4310/98/$19.00 PII: S1357-4310(97)01166-0
Almost every aspect of a cell’s growth, differen-tiation, function (dysfunction) and even death, iscontrolled primarily by the transcription of geneswhose protein products contribute to these pro-cesses. In order to understand the basis of thesecellular events it is therefore essential to knowwhere and when genes are expressed and, moreimportantly, how the complement of genes ex-pressed by cells under one set of conditions differsfrom the same or a similar population of cells inother circumstances. In other words, which genesare differentially expressed and how do the pro-teins they encode control or alter the phenotype ofthe cell? For years the most widely used means ofperforming a comparative expression analysiswas a technique called subtractive hybridization.Although applied with some success, the method-ology was long, laborious and not generally sensi-tive enough to get at the interesting, low copy num-ber genes.
When, in 1992, the technique of differential dis-play was published by Liang and Pardee, it offered a rapid, sensitive and inexpensive way toidentify many, if not all, of the genes that are dif-ferentially expressed between two or more popu-lations of cells, using relatively simple methodol-ogy. Differential display is based on the use ofshort PCR primers, ten bases in length, that an-neal arbitrarily to cDNA species that contain acomplementary sequence. In combination withanchored T-primers that prime the PCR reactionfrom the 3' end of polyadenylated transcripts, itprovides a means of amplifying 3' fragments froma range of mRNAs.Separation of the labelled PCRproducts by acrylamide gel electrophoresis givesa series of bands, each representing a fragment ofa gene expressed in those cells. If a band is am-plified from one population but not the other, it islikely to be a fragment of a differently expressedgene, and therefore of interest and worthy of fur-ther characterization.
Since the publication of this technique, differ-ential display has captured the imagination of
many biologists and has now been successfullyapplied for the isolation of differentially expressedgenes in a diverse range of biological systems. Intheory, differential display is relatively easy to per-form, but in practice, many aspects of the tech-nique can and do cause problems.This book is de-signed to help those intent on using this andrelated methodologies. It comprises a collection ofworks from various laboratories each with theirown experience of, modifications to, and appli-cations for, differential display. The first part of thebook is dedicated to the basic principles of themethodology, together with chapters on the re-lated method of RNA arbitrary primed PCR (RAP-PCR) and a study that has compared differentialdisplay with subtractive hybridization. This sectionalso includes details of advances in the methodol-ogy, such as the use of fluorescence to label am-plified fragments and a number of chapters outlin-ing protocols to confirm differential expression (asfalse positives are not uncommon) and the cloningand sequencing of differentially expressed genefragments.Part two of the book contains two chap-ters that detail work that has modified the ap-proach in order to search for family members ofpreviously characterized genes, while the remain-der of the book concentrates on the application ofthe technique to study differentially expressedgenes in a diverse range of biological systems; in-ducible genes; and genes differentially expressedduring development, differentiation or disease. Assuch, the book covers a wide spectrum of appli-cations, which rightly reflects the technique’s al-most universal applicability to the examination ofdifferential gene expression. In common with othervolumes in the popular ‘Methods in MolecularBiology’ series, each chapter starts with a short in-troduction providing a background to the work, fol-lowed by detailed method sections giving the kindof essential information almost never found in pa-pers. Each chapter is illustrated with examples ofthe results and ends with helpful notes and refer-ences.
There are now a number of alternative strat-egies that could be employed by those wishing toexamine the regulation of known or unknowngenes. Expressed sequence tag (EST) sequenc-ing and serial analysis of gene expression (SAGE)rely on sequencing large numbers of gene frag-ments and then analysing how often a sequenceis present in one population but not the other, whilethe Affymetrix chip and cDNA array systems relyon the hybridization of RNAs labelled with a fluo-rochrome to arrays of oligonucleotides or cDNAscomplementary to hundreds or thousands ofgenes. Although these techniques also provide ahandle on differentially expressed genes they areeither very new and therefore poorly character-ized, or expensive to set up and therefore currentlybeyond the pocket of many laboratories. As a re-sult, differential display remains, at least for thetime being, the most popular and affordable sys-tem to study and isolate differentially expressedgenes. For any would-be gene hunter thinking ofor already practising differential display, this bookwill provide an invaluable and long over-due guideto the technique.
Tom Freeman PhDExpression Group Leader
Human Genetics Group, The Sanger Centre,Wellcome Trust Genome Campus, Hinxton,
Cambridge, UK CB10 1SA.
Tel: +44 1223 494907Fax: +44 1223 494919
e-mail: [email protected]
Differential Display Methods and Protocols edited by Peng Liang and Arther B. Pardee,Humana Press, 1997. $69.50 (paperback)/$99.50 (hardback) (xiv + 304 pages) ISBN 0 896 0340
Detecting differences in gene expression