This kit is designed for rapid isolation of total RNA from different types of tissue and cultured cells. This type of RNA extraction is environmentally friendly, does not require phenol or other hazardous solutions and therefore, can be performed very conveniently on the lab bench without the need for a fume hood. It avoids toxic organic solvents by utilizing a rapid desalting process to remove contaminating macromolecules. The purified RNA can be used subsequently in many applications including hybridization, RNase protection and RT-PCR. According to the kit manual, RNA can be extracted from a variety of sources, including bovine liver, human HL-60 tissue culture cells (0.5-1 x 10
6 mammalian cells), human whole blood and plasma, saliva, corn and geranium leaf, yeast;
E. coli, and lambda phage, even from formalin formalin-fixed, paraffin-embedded (FFPE) tissues.
The first step of RNA extraction is the lysis of the tissue or pelleted cells with Tissue and Cell Lysis Solution with added 50 µg Proteinase K. According the kit's instructions, you should choose the volume of the Lysis Solution for your specific cell type. The protocols are different depending upon the specific cell type. In my case, I purified total RNA from 2.5x106 human PBMCs. Thus, I added 300 µl of Tissue and Cell Lysis Solution containing the Proteinase K and mixed thoroughly. Then, samples were incubated at 65°C for 15 minutes and vortexed every 5 minutes. After that, I placed the sample on ice for 3-5 min and then proceeded with RNA precipitation. 175 µl of Protein Precipitation Reagent was added to 300 µl of lysed sample and vortexed. Debris was pelleted by centrifugation for 10 minutes at 10,000 x g in a microcentrifuge. Then, supernatant was transferred into clean tube and 500 µl of isopropanol was added to the supernatant. The tube was inverted several times to mix the solutions. Total RNA was collected by centrifugation at 4°C for 10 or 30 minutes in a microcentrifuge. Then isopropanol was discarded and I proceeded to remove contaminating DNA, because I needed this RNA for subsequent in vitro transcription.
To remove DNA, 5 µl of DNaseI (provided) was added into 195 µl of DNase I buffer.
I incubated the total RNA for 20 min to assure complete DNA removal. I then proceeded to a second RNA precipitation: again, I added 200 µl Lysis Solution and vortexed for 5 seconds, then 200 µl of MPC Protein Precipitation Reagent was added and the tube was placed on ice, 3-5 min. The debris was pelleted by centrifugation for 10 minutes at 10,000 x g in a microcentrifuge.
The supernatant containing the RNA was transferred into a clean microcentrifuge tube and the pellet was discarded. In order to recover the RNA, 500 µl of isopropanol was added to the supernatant. The resulting purified RNA was centrifuged at 4°C for 10 minutes in a microcentrifuge, washed twice with 75% ethanol. RNA was recovered in 15 µl of double distilled water. I checked the quality of the precipitated RNA by running it on a 2.5% agarose gel and its concentration by using a capillary spectrophotometer. Unfortunately, both tests showed bad results. I obtained only smeary degraded RNA on the gel and its concentration and quality was very low, according the spectrophotometer. In parallel to RNA extraction using the MasterPure™ RNA Purification Kit, I performed total RNA extraction from the same number of PBMCs (2.5X106) using a traditional phenol/chloroform method. I obtained 2-3 ìg of good quality total RNA using traditional method. I contacted the Technical Support team and after sending them all of my data, they returned to us the cost of the kit.
Dr. Nataly Urshanski
Neuroimmunology Laboratory Manager
Department of Neurology
Sourasky Tel-Aviv Medical Research Center
Israel
Epicentre responds:
We would like to respond to Dr. Urshanski’s assertion that “RNA extraction is not successful from cell cultures.” Dr. Urshanski had contacted our distributor in Israel after purchasing the kit; however, she declined technical support offered by our distributor in Israel who worked with our scientists. Instead, she requested a refund of the purchase price. The results that Dr. Urshanski observed with cell culture samples are not typical in our experience, as well as that of other customers.
The MasterPure RNA kit has been used successfully with both adherent and suspension cell cultures (Fig. 1). The data show that the RNA isolated from these cell lines is intact and not degraded, as the review implies. Other customers have used the kit with good results from cultured cells, including cell types used by the reviewer.1-4
For those customers experiencing problems with cultured cells, we offer the following suggestions:
1. Wash cells with sterile PBS or an equivalent buffer to remove serum proteins and culture media from cells before cell lysis.
2. Adherent cells can be lysed directly in culture flasks or plates without the use of trypsin to release them. Add Tissue and Cell Lysis Solution directly to washed cells.
3. Cells that were collected by centrifugation prior to purification should be resuspended in a small amount of PBS before the addition of lysis solution to ensure uniform lysis.
4. Work quickly from washing cells to lysing cells to preserve expression patterns.
5. Maintain an RNase-free work environment.
We also encourage our international customers to contact our technical support scientists directly by e-mail at techhelp@epibio.com.
Fig. 1. Full-length total cellular RNA purified from five different cultured cell lines with the MasterPure™ RNA Purification Kit. Both adherent and suspension cell cultures were purified as per the protocol. 200 ng of each RNA preparation was separated by denaturing formaldehyde agarose gel electrophoresis and visualized with SYBR® Gold. Lane M, RNA Marker; RNA purified from cell lines as indicated: Lane 1, HeLa; Lane 2, Ramos; Lane 3, BDCM; Lane 4, NIH3T3; Lane 5, NRK.
References
1. Chiosa, S. et al. Overexpression of Dicer in Precursor Lesions of Lung Adenocarcinoma. Cancer Res (2007) 67:2345-2350.
2. Chao, H. et al. Phenotype correction of hemophilia A mice by spliceosome-mediated RNA trans-splicing. Nature Medicine (2003) 9:1015-1019.
3. Phelan, P. E. et al. Characterization of Snakehead Rhabdovirus Infection in Zebrafish (Danio rerio). J Virol (2005) 79:1842-1852.
4. Kramer, M. F. et al. Latent Herpes Simplex Virus Infection of Sensory Neurons Alters Neuronal Gene Expression. J Virol (2003) 77:9533-9541.