Expression Profiling of Single Mammalian Cells – Small is Beautiful

Abstract

<jats:p>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.</jats:p>

Differential gene expression of human stem progenitor cells derived from early stages of in utero human hematopoiesis

“…15 However, one of the major difficulties in performing SSH in enriched HSPCs is the inability to obtain a sufficient number of cells to generate an adequate amount of cDNA using standard procedures. We overcame this limitation by amplification of first-strand cDNA, as was done in previous studies, 16,22,23 in which the relative abundance of the primary transcripts is maintained during the amplification process. Given the distinct characteristics of FB-HSPC and its corresponding population of adult MPB-HSPC with identical cell surface phenotype, we used amplified cDNA of MPB-HSPCs as driver in SSH to remove genes that are equally expressed in both populations and enrich for transcripts that are differentially expressed in tester template generated from FB-HSPCs.…”

Clinical quantitation of immune signature in follicular lymphoma by RT-PCR–based gene expression profiling

“…As such, it is better suited for measurement in clinical practice, while also focusing on diagnostically relevant Indicator genes. This approach thereby enables gene signatures to be detected within very small amounts of pathological material, 15 and we have recently demonstrated its utility in diffuse large B-cell lymphoma and follicular lymphoma. 11 In this study, we have used the same approach to measure immune gene signatures in follicular lymphoma.…”

Gene expression profiling of the developing mouse kidney and embryo

“…Embryonic and newborn mouse tissues were dissected from the embryo, and RNA was isolated from the tissues. Two microgrammes of RNA was reverse transcribed and globally amplified by polyAPCR, which preserves the relative abundance of RNA in the starting sample (Al-Taher et al 2000;Brady 2000;Iscove et al 2002). The polyAcDNA from each individual tissue sample was assessed for Gapdh expression by real-time qPCR, and the relative expression levels for individual tissues at different gestation stages are shown in the graph.…”

“…This procedure amplifies all polyadenylated RNA in a given sample. The cDNA collection thus produced preserves the relative abundance of the mRNAs present in the original sample (Al-Taher et al 2000;Brady 2000;Iscove et al 2002). Two microlitre of RNA from explanted embryonic and newborn tissues were reversed transcribed as previously described (Brady and Iscove 1993).…”