Nuclear expressed sequence tag (NEST) analysis: A novel means to study transcription through amplification of nuclear RNA

We describe a novel concept and corresponding methods for the analysis of transcription in higher plant cells. The concept is that an examination of the presence of different polyadenylated transcripts within isolated nuclei reflects the state of gene expression at a given moment more precisely than do conventional techniques using total cellular mRNA. The methods involve isolation of polyadenylated nuclear transcripts from flow-sorted nuclei, reverse transcription, amplification using the polymerase chain reaction, and analysis of the products through gel electrophoresis and sequencing. By using specific primers, we have demonstrated detection of selected gene products in nuclei from transgenic plants. We also employed a technique for analysis of individual transcripts based on the length polymorphisms of restriction fragments derived from their 3' ends. Because the technique does not require a priori knowledge of the analyzed sequences, it is suitable for displaying the complete spectra of RNA transcripts present in nuclei at the moment of their isolation. These fragments can be easily isolated and sequenced and the sequence information used for assignment of putative function of corresponding genes. These techniques have been used to identify leaf-, root- , and cell cycle-specific transcripts. In principle, they should be applicable to the tissues of any eukaryotic species that contain transcriptionally active nuclei.