Abstract
Increasingly mRNA expression patterns established using a variety of molecular technologies such as cDNA microarrays, SAGE and cDNA display are being used to identify potential regulatory genes and as a means of providing valuable insights into the biological status of the starting sample. Until recently, the application of these techniques has been limited to mRNA isolated from millions or, at very best, several thousand cells thereby restricting the study of small samples and complex tissues. To overcome this limitation a variety of amplification approaches have been developed which are capable of broadly evaluating mRNA expression patterns in single cells. This review will describe approaches that have been employed to examine global gene expression patterns either in small numbers of cells or, wherever possible, in actual isolated single cells. The first half of the review will summarize the technical aspects of methods developed for single-cell analysis and the latter half of the review will describe the areas of biological research that have benefited from single-cell expression analysis.
Highlights
Following enormous advances in the area of genomics and the complete sequencing of the human genome, the current challenge to biologists is to learn how the products of the 30 000±150 000 identi®ed genes interact to produce the complexity exhibited by higher eukaryotes
Recognition of the value of the examination of expression patterns led to the development of a plethora of more advanced technologies, such as cDNA microarrays [22], SAGE [58] and cDNA display [37] aimed at the simultaneous measurement of tens to several thousand genes in the target samples
In the original singlecell mRNA phenotyping method, following total RNA isolation, cDNA was prepared in a reverse transcriptase (RT) reaction using an oligo dT primer and separate gene-speci®c PCRs were carried out on aliquots of the total cDNA [45]
Summary
Following enormous advances in the area of genomics and the complete sequencing of the human genome, the current challenge to biologists is to learn how the products of the 30 000±150 000 identi®ed genes interact to produce the complexity exhibited by higher eukaryotes. In the late 1980s, Rapopolee and colleagues described a protocol known assingle-cell mRNA phenotyping', which was developed for the analysis of multiple genes (10 or more) in small samples, including single cells [44,45]. In the original singlecell mRNA phenotyping method, following total RNA isolation, cDNA was prepared in a reverse transcriptase (RT) reaction using an oligo dT primer and separate gene-speci®c PCRs were carried out on aliquots of the total cDNA [45].
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