RNA yield increases with labeling time. a Plot of total yield of RNA recovered in nanograms per OD600 unit of cells against labeling time in minutes. During the first five minutes of labeling, yield increases linearly with labeling time (R2 = 0.985). Some RNA is recovered when 4-thiouracil (4tU) is not added to the culture (0 min). This is essentially background RNA that non-specifically bound to the magnetic beads during the isolation procedure. The horizontal dashed line indicates the level of background for this experiment. The actual amounts of RNA recovered from 600 ml culture (average of two experiments; R2 = 0.960) were: 0.50 μg (0 min), 0.77 μg (1 min), 1.16 μg (1.5 min), 3.30 μg (5 min) and 4.52 μg (10 min). b Agilent Bioanalyzer trace demonstrating the qualitative differences between the different fractions produced during the isolation of newly synthesized RNA. The left panel displays the results when no 4tU is added while the right panel shows 2.5 min labeling. The nonspecific background can be seen to be different from the total RNA comprising mostly short fragments. tRNA transfer RNA. 25S, 18S and 5S indicate mature ribosomal RNA species.