Extracting High Quality RNA from Bone in a Single Step. (A) Isolated liver RNA was incubated with RNase free beads (lane 1) or untreated beads (lane 2) provided by the manufacturer (Next Advance) for four hours before analyzing the 18S and 28S rRNA bands using agarose gel chromatography. Subsequent experiments with the Bullet Blender were carried out using untreated beads. (B) Bone RNA was homogenized in near freezing conditions using the Bullet Blender centrifuge (lanes 2-4) or a Polytron (lanes 5-6). The results are compared to bone RNA isolated using standard homogenization conditions (lane 1) and intact liver RNA that was previously isolated (control). (C) The RNA Integrity Number (RIN) for RNA homogenized in near freezing conditions was determined using the Agilent RNA 6000 Nano LabChip Kit and the Agilent 2100 Bioanalyzer. RNA with a RIN = 7 or greater is suitable for microarray analysis of gene expression. (D) The maximum RIN for the two-step approach (lane 2) is compared to the maximum RIN obtained in the one-step approach (lane 3) and each RIN is compared to the RIN of the degraded RNA sample (lane 1) shown in the agarose gel in lane 1, Figure 2B. (E) The electropherograms associated with the samples shown in (D).
Carter et al. BMC Biotechnology 2012 12:5 doi:10.1186/1472-6750-12-5