Expression of the imprinted tumour‐suppressor gene H19 is tightly regulated during normal haematopoiesis and is reduced in haematopoietic precursors of patients with the myeloproliferative disease polycythaemia vera

Núñez, César; Bashein, Abdulla; Brunet, C. L.; Hoyland, Judith A.; Freemont, Anthony J.; Buckle, Anne Marie; Murphy, Clare; Cross, Michael; Lucas, Guy; Bostock, Victoria; Brady, Gerard

doi:10.1002/(sici)1096-9896(200001)190:1<61::aid-path502>3.0.co;2-#

Cited by 18 publications

References 36 publications

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Global cDNA Amplification Combined with Real-Time RT–PCR: Accurate Quantification of Multiple Human Potassium Channel Genes at the Single Cell Level

Al-Taher

Bashein

Nolan

et al. 2000

Yeast

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We have developed a sensitive quantitative RT–PCR procedure suitable for the analysis of small samples, including single cells, and have used it to measure levels of potassium channel mRNAs in a panel of human tissues and small numbers of cells grown in culture. The method involves an initial global amplification of cDNA derived from all added polyadenylated mRNA followed by quantitative RT–PCR of individual genes using specific primers. In order to facilitate rapid and accurate processing of samples, we have adapted the approach to allow use ofTaqMan™real-time quantitative PCR. We demonstrate that the approach represents a major improvement over existing conventional and real-time quantitative PCR approaches, since it can be applied to samples equivalent to a single cell, is able to accurately measure expression levels equivalent to less than 1/100th copy/cell (one specific cDNA molecule present amongst108total cDNA molecules). Furthermore, since the initial step involves a global amplification of all expressed genes, a permanent cDNA archive is generated from each sample, which can be regenerated indefinitely for further expression analysis.

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Global cDNA Amplification Combined with Real-Time RT–PCR: Accurate Quantification of Multiple Human Potassium Channel Genes at the Single Cell Level

Al-Taher

Bashein

Nolan

et al. 2000

Yeast

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Expression Profiling of Single Mammalian Cells – Small is Beautiful

Brady

2000

Yeast

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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.

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Noncoding Regulatory RNAs in Hematopoiesis

Jeong

Goodell

2016

Current Topics in Developmental Biology

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Expression of the imprinted tumour‐suppressor gene H19 is tightly regulated during normal haematopoiesis and is reduced in haematopoietic precursors of patients with the myeloproliferative disease polycythaemia vera

Cited by 18 publications

References 36 publications

Global cDNA Amplification Combined with Real-Time RT–PCR: Accurate Quantification of Multiple Human Potassium Channel Genes at the Single Cell Level

Global cDNA Amplification Combined with Real-Time RT–PCR: Accurate Quantification of Multiple Human Potassium Channel Genes at the Single Cell Level

Expression Profiling of Single Mammalian Cells – Small is Beautiful

Noncoding Regulatory RNAs in Hematopoiesis

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