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EPICENTRE Forum 5 (2) The MasterPure™ Complete DNA and RNA Purification Kit: A Flexible System for Rapid Isolation of DNA and RNA from a Wide Variety of Sources Judith T. Schanke and John Watson, EPICENTRE Technologies Introduction Pure, high molecular weight genomic DNA and intact RNA are used for numerous applications in both molecular diagnostic and basic research laboratories. EPICENTRE's new MasterPure™ Complete DNA and RNA Purification Kit can be used to isolate purified genomic DNA, RNA, or total nucleic acid (both DNA and RNA) in minimal time without using resins, columns, or hazardous organic solutions. Researchers can routinely prepare genomic DNA or RNA in approximately 30 minutes. Highly purified, total cellular RNA can be prepared in less than one hour. Reagents and protocols are provided for easy, efficient extraction of DNA and RNA from limited amounts of starting material and from a wide range of sources including frozen or fresh mammalian tissue, plant tissue, cultured cells, blood or serum samples, mouse tails, bacteria, yeast, and paraffin-embedded tissues. In addition, the MasterPure Complete Kit protocol can be easily adjusted for large or small sample sizes. The basic MasterPure procedure for isolating genomic DNA, RNA, and total nucleic acid is outlined in Figure 1. The method utilizes a rapid desalting process to remove contaminating macromolecules.1 Typically, the cells are lysed, proteins are removed, and the total nucleic acid is efficiently precipitated, resulting in a visible pellet even when using a limited amount of starting material. To isolate purified DNA, the sample is treated with RNase A before the protein precipitation step. To isolate purified RNA, the precipitated total nucleic acid sample is treated with DNase I, the DNase I is removed, and the pure RNA is precipitated again.
In this study, we demonstrate the purification of high molecular weight DNA, intact RNA, and total nucleic acid from a variety of sources using the MasterPure Complete DNA and RNA Purification Kit. We show that genomic DNA purified from bovine liver using the MasterPure Complete Kit can be easily PCR-amplified. We also use the MasterAmp™ RT-PCR Kit to amplify a message from total cellular RNA isolated from bovine liver using the MasterPure Complete Kit. Methods and Results Consistent production of pure, high molecular weight genomic DNA, total nucleic acid, and RNA Fifteen separate 2 mg samples of frozen bovine liver were used to prepare DNA, total nucleic acid (containing both DNA and RNA), or RNA using the MasterPure Complete DNA and RNA Purification Kit. Each 2 mg sample was suspended in 300 µl of Tissue and Cell Lysis Buffer and the tissue was homogenized briefly. Proteinase K was added (2 µl of a 25 mg/ml stock) and the samples were incubated for 15 minutes at 60°C, with vortexing after 10 minutes. Purified genomic DNA was isolated from five of the samples. These samples were treated with 3 µg of RNase A for 30 minutes at 37°C. Then, 150 µl of Protein Precipitation Reagent were added and the samples were mixed by vortexing for 10 seconds. The samples were centrifuged for 10 minutes at 4°C in a microcentrifuge. The DNA-containing supernatants were transferred to clean tubes and 500 µl of isopropanol were added to each sample. The tubes were inverted 30 times and then centrifuged for 10 minutes at 4°C in a microcentrifuge. The isopropanol was decanted and the easily visible pellets were washed twice with 70% ethanol. The DNA pellets were then resuspended in 50 µl of TE Buffer. Total nucleic acid was prepared from five of the 2 mg bovine liver samples using the same procedure described for DNA isolation, except the RNase A treatment was omitted. This procedure results in the isolation of both high molecular weight genomic DNA, as well as ribosomal and messenger RNA. To obtain pure cellular RNA, five of the 2 mg bovine liver samples were also processed as described above, without the RNase A treatment. The nucleic acid pellet was then resuspended and treated with 1 unit of DNase I for 30 minutes at 37°C. The protein precipitation steps were repeated and the RNA pellets were resuspended in 50 µl of TE Buffer. Analysis of the DNA, total nucleic acid, and RNA samples Fifteen microliters of each sample were separated by agarose gel electrophoresis,
stained with ethidium bromide, and visualized by UV illumination. The
high molecular weight genomic DNA, total nucleic acid isolations, and
purified RNA from bovine liver are shown in Figure 2.
Isolation of DNA and RNA from a wide variety of cell sources The protocols described above were used to isolate DNA, total nucleic
acid, or RNA from different cell sources. The isolated nucleic acids
are shown in Figure 3. DNA, total nucleic acid, and RNA were isolated
from the human tissue culture cell line HL60 (Panel A) and E. coli (Panel
B). Total nucleic acid isolated from the Gram-positive bacterium Staphylococcus
aureus (Panel C), a mouse tail snip (Panel D), and maize (Panel E)
is also shown. Saccharomyces cerevisiae RNA is shown in Panel
F. High molecular weight genomic DNA is seen in these figures, as well
as prominent ribosomal RNA bands. The OD260/280 of all DNA
and RNA samples isolated ranged from 1.8 to 2.0. The MasterPure Complete
DNA and RNA Purification Kit consistently produced high molecular weight,
pure nucleic acid from all cell sources tested. 
Amplification of the melanocortin receptor 1 gene and the prolactin receptor message from bovine liver DNA and RNA To demonstrate that DNA isolated from bovine liver using the MasterPure
Complete Kit could be easily amplified by PCR, we amplified a region
of the melanocortin receptor 1 (MC1R) gene. The primers used to amplify
the 131 bp region of MC1R were: 5'-GCTGTGTCTGACTTGCTGGT-3' and 5'-ATGAGCACGTCGATGACATT-3'.2 One
microliter of the bovine liver DNA isolated above was included in a 50 µl
reaction containing MasterAmp PCR Optimization Kit PreMixes E and H (containing
2.5 mM MgCl2, 2X or 3X MasterAmp PCR Enhancer (with betaine*),
and 200 µM each dNTP) (EPICENTRE Technologies), 12.5 pmoles of
each primer, and 1.25 units of MasterAmp Taq DNA Polymerase (EPICENTRE
Technologies). The reactions were cycled 30 times as follows: 92°C
for 30 seconds, 60°C for 1 minute, and 72°C for 1 minute. Ten
percent (5 µl) of each sample was separated by agarose gel electrophoresis,
stained with ethidium bromide, and visualized by UV illumination. Figure
4A shows the specific, high-yield amplification of the 131 bp region
of the MC1R gene from bovine liver DNA isolated with the MasterPure Complete
Kit.
To demonstrate that the total cellular RNA isolated with the MasterPure Complete Kit was a good template for RT-PCR, we amplified a 643 bp region of the prolactin receptor (PRLR) message using the MasterAmp RT-PCR Kit (EPICENTRE Technologies). The PRLR primers were: 5'-GCTGTGTCTGACTTGCTGGT-3' and 5'-ATGAGCACGTCGATGACATT-3'.3 The reaction contained 5 µl of purified RNA template (10% of the sample isolated above), 1X RT-PCR buffer, 3 mM MgCl2, 3X MasterAmp PCR Enhancer (with betaine), 0.5 mM MnSO4, 400 µM each dNTP, 12.5 pmoles of each primer, and 2.5 units of RetroAmp™ RT DNA Polymerase in a total volume of 50 µl. The RNA was reverse transcribed at 60°C for 20 minutes. Then, 30 cycles were performed as follows: 92°C for 40 seconds, 60°C for 1 minute, and 72°C for 1 minute. Ten percent (5 µl) of each sample was separated by agarose gel electrophoresis, stained with ethidium bromide, and visualized by UV illumination. The 643 bp RT-PCR amplification product of the bovine PRLR message is shown in Figure 4B. Summary The MasterPure Complete DNA and RNA Purification Kit can be used to isolate pure, high molecular weight DNA, total cellular RNA, or total nucleic acid from many cell sources, even with a limited amount of starting material. We have demonstrated the purification of high-quality DNA and RNA from tissues as diverse as yeast, E. coli, plant tissue, and mammalian tissues. The DNA and RNA purified using the MasterPure Kit was suitable for use as a template for PCR amplification.
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