Identification of Newly Transcribed RNA

Abstract

AbstractNewly transcribed RNA can be identified using the nuclear runoff transcription assay. Isolated nuclei, free of membranes and cytoplasmic debris, are required for the assay. Cell lysis that does not allow the isolation of nuclei free of cell membranes and cytoplasmic material often results in poor incorporation of 32P‐labeled UTP into nascent transcripts. Although there is no way to predict what cell types present this problem, many adherent cell lines and lymphocytes isolated from murine spleen or thymus do. Interestingly, very few nonadherent cell lines have posed this problem. As described in this unit, isolating nuclei by detergent lysis of cells works well for many tissue culture cell lines but may not be appropriate for all cell lines and many tissues. Protocols for detergent lysis and Dounce homogenization or cell lysis in an isoosmotic solution and centrifugation through a sucrose cushion are described along with a for the preparation of the cDNA nitrocellulose filter strips that are used to detect the presence of specific transcripts in the nuclear runoff transcription assay.