Authors: Nasrin Shojaie, Mahmood S. Ghaffari & Zahra Safari
Simultaneous investigation of DNA and RNA is often a necessity in genetic manipulation and biological researches. Besides, most of the traditional procedures devised for RNA isolation have some difficulties associated with RNase activity. Therefore, this protocol presents a safer process to extract high purity RNA in shorter time.
The protocol describes a simple and less time-consuming method for nucleic acid extraction from animal cell culture with high purity. This process could prove useful for simultaneous study of DNA and RNA extracted from a unique sample. After adding isopropanol to cell lysate, RNA precipitation occurs and simultaneously, DNA containing suspension is isolated, in the following step, RNA and DNA are separately purified by absolute ethanol. Using the present protocol, the yield of total extraction is proportional to the amount of the starting sample; however, the minimum number of the cells with a favorable qualification is 2×10e6. Moreover, all solutions used in this protocol could be prepared with autoclaved ddH2O diethyl pyrocarbonate (DEPC) free.
For mammalian cells grown in monolayer:
Steps 2 to 6: 30 min Step 7: 60 min Step 8: 60 min
Step 3: Turbidity in mixture Cell debris - After transferring lysate into a 1.5 ml microtube, if you see any turbidity, centrifuge mixture at 10,000g at 4 °C for 5 min and remove cell debris.
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).
Source:Protocol Exchange (2014) Originally published online 16 September 2014