Evaluation of lysis buffers. (A) Determination of lysis efficiency. Each bar indicate relative yield of Ins2 using a single pancreatic islet (~1000 cells) as starting material. Each islet was treated with indicated concentrations of either NP-40, with and without proteinase K (Prot K) treatment, or guanidine thiocyanate (GuSCN). Only lysis with 0.5 M GuSCN had a significant effect (>500-fold increase) compared to the water control (p < 0.001, n = 3). The value of the control was arbitrarily set to 1. Similar results were obtained for Gcg and Rps29. (B) Effect of lysis buffers on RT reaction yield. Identical amounts of purified islet total RNA was used as starting material. Relative yields of five genes were analysed: Ins1, Gcg, Sst, Gapdh and Rps29. Increasing concentrations of GuSCN was added to the RT reaction. There is a significant difference for all genes between control and both 40 mM and 120 mM (p < 0.05) but not 80 mM. The expression value was arbitrarily set to 1 for all genes at 0 mM GuSCN. Values are mean ± SEM for three separate experiments. The experiments in (A) and (B) were carried out without the presence of RNase inhibitor.
Bengtsson et al. BMC Molecular Biology 2008 9:63 doi:10.1186/1471-2199-9-63