Novel method for studying mRNA phenotypes in single or small numbers of cells

作者: Daniel A. Rappolee , Alice Wang , David Mark , Zena Werb

DOI: 10.1002/JCB.240390102

关键词:

摘要: Biological processes, such as growth control, are often governed by biochemical steps involving mRNA transcripts that short-lived and have a low copy number. Furthermore, the cells involved in these processes available numbers from vivo sources. We now report method is superior to situ hybridization, RNA blot analysis, nuclease protection assay for study of short-lived, low-copy-number transcripts. The consists microprocedure isolating one few thousand two coupled enzymatic steps: reverse transcription whole cellular RNA, followed amplification cDNA specifically primed polymerase chain reaction give specific fragments can be visualized on agarose gels ethidium bromide staining. With this we detected actin single cell, or less than 100 cRNA molecules, quantified differences concentrations threefold. products divided reaction, several species assayed simultaneously. Therefore, call single-cell phenotyping. This technique applicable analysis factor culture vivo.

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