Figure 4

RNA degradation is reduced during lysis by grinding. A, Strains RJY358 (wt, untagged strain), RJY929 (Nrp1p-TAP) and RJY933 (Pbp2p-TAP) were manually ground in liquid nitrogen. Samples from crude lysate were phenol extracted and 8 μg of the extracted RNA were loaded onto 1.2% agarose-formaldehyde gels and blotted onto nylon membranes. 8 μg of total RNA (P/C lysis RNA) from strains RJY358 (wt, untagged strain), RJY929 (Nrp1p-TAP) and RJY933 (Pbp2p-TAP) prepared with a phenol extraction method [18] were loaded in parallel as control for intact RNAs. B, Strains RJY358 (wt, untagged strain), RJY929 (Nrp1p-TAP) and RJY933 (Pbp2p-TAP) were ground in a motor-driven mortar in the presence of dry ice. Samples from crude lysate, supernatant of 20000 × g (S20) spin, and pellet of 200000 × g (P200) spin were phenol extracted and 8 μg of the extracted RNA were loaded onto 1.2% agarose-formaldehyde gels and blotted onto nylon membranes. As control for RNA quality, RNA samples from independently lysed cells were loaded on the same gel. The positions of the 18S and 25S ribosomal RNAs in the methylene blue staining and the PDA1 mRNA hybridization signal are indicated.