The critical step in this protocol is formulation of the extraction buffer. The composition of this buffer is Laemmli buffer Bromophenol Blue free, that can completely break the links between bio-macromolecules (DNA, RNA and proteins), decrease the possibility for creation of new connections between them and specially stabilize the unfolded proteins by 2-ME and SDS. In addition, SDS and Glycerol aid lipids dissolve in the buffer and alcohols. The precipitation of nucleic acids, particularly RNA, occurs in the presence of isopropanol. Finally, ethanol plays an effective role in obtaining a high-quality nucleic acid extraction . In fact, in all the steps of this procedure RNase is inactive, thus RNA can be intact in the presence of extraction buffer, isopropanol and ethanol.
We examined this protocol for 52 samples of six cell lines (PLC/PRF/5, MDA-MB231, MCF7, NIH-3T3, SKNMC and HepG2). Data analysis was performed using one sample t-test, SPSS software version 17 (95% CI). The results showed that, for DNA concentration, the mean ± SE for ratio of OD260/OD280 and OD260/230 were 1464.80±185.63 ng/µl, 1.80±0.007 and 2.08±0.025, respectively. While for RNA concentration, the ratio of OD260/OD280 and OD260/230 were 627.35±56.86 ng/µl, 1.92±0.03 and 1.70±0.04, respectively (Fig. 1a). It was previously reported that the accepted range for OD260/OD280 and OD260/230 ratios are more than 1.8 and greater than 1.5, respectively [2-3].
Furthermore, this protocol was applied to extract DNA in order to do DNA fragmentation assay (Fig. 1b). Also, the quality of the extracted RNA was examined by evaluating the expression of a variety of genes, including p50, HIF-1α, Lgr5 and β-catenin using RT-PCR (Fig. 2).