R. Strand et al., THE CHOICE OF RESUSPENSION MEDIUM FOR ISOLATED RAT-LIVER NUCLEI - EFFECTS ON NUCLEAR MORPHOLOGY AND IN-VITRO TRANSCRIPTION, Molecular and cellular biochemistry, 139(2), 1994, pp. 149-157
Standard protocols for in vitro transcription assay (nuclear run-off)
include 10-40% (v/v) glycerol (of various ionic strength) in the mediu
m used for resuspension/storage of the isolated nuclei. In the present
work the morphological and functional properties of nuclei isolated f
rom rat liver have been studied as a function of the content of glycer
ol, sucrose and inorganic ions (K+ and Mg2+) in the resuspension mediu
m. In contrast to earlier reports, glycerol was found not to be essent
ial to maintain morphological integrity and RNA polymerase activity in
frozen/stored nuclei. Nuclear pellets, resuspended and stored in isoo
smotic sucrose media, were found to give morphologically intact and tr
anscriptionally active nuclei. Furthermore, these nuclei displayed a h
igher specific hybridization signal for the differentially expressed g
enes encoding peroxisomal beta-oxidation enzymes, relative to the tota
l RNA synthesis, than nuclei resuspended and stored in a hyperosmotic
glycerol-containing medium. The concentrations of inorganic ions were
also found to affect nuclear morphology. Flow cytometry indicated DNA
leakage from nuclei at insufficient concentrations of K+ and Mg2+, and
high ionic strength favoured aggregation and disintegration of nuclei
. Our findings indicate that quantitative results from nuclear run-off
experiments should be interpreted with caution until the process of t
ranscription in isolated nuclei is better understood.