RNA localization by nuclear/cytoplasmic fractionation of cells.

After the specified treatments, cells were trypsinized, counted, and divided into two equal aliquots (one for total RNA isolation and the second for nuclear and cytoplasmic fractionation). Both cell aliquots were lysed on ice in cell lysis buffer for 10 to 20 min. For nuclear/cytoplasmic fractionation, the lysate was centrifuged at 14,000 rpm to collect the nuclear pellet and soluble fraction. The fractions as well as the lysate for total RNA isolation were treated with buffer G (RNA isolation kit cat no. 25501; Active Motif) in 70% ethanol, loaded on to a spin column, and subjected to centrifugation; the retained precipitate was washed with ethanol one time and was eluted in RNase-free water. cDNAs were prepared from various RNA preparations, and qRT-PCR was performed (as mentioned above). mRNAs from nuclear and cytoplasmic fractions were normalized to total RNA isolated from an identical number of cells.