Get Better Viral RNA Detection from 10-fold Less Sample
To rigorously test the MagMAX Viral RNA Isolation Kit, we sought the assistance of the National Veterinary Services Laboraties (NVSL), a subsidiary of the Animal and Plant Health Inspection Service (a division of the USDA). NVSL is charged with providing animal disease testing for Veterinary Services and testing of domestic and foreign diagnostic specimens for animal diseases. Currently NVSL uses another manufacturers RNA isolation product to obtain RNA from biological fluids for their validated viral testing process. When they compared this validated system to Ambions MagMAX Viral RNA Isolation Kit, they found that they could get similar, and often better results using 10-fold less sample with the MagMAX Kit. The experiment shown in Figure 1 is a comparison of Ct values in a real-time qRT-PCR assay for the Exotic Newcastle Disease (END) virus. Cloacal or tracheal swab samples were collected from poultry infected with the virus, and RNA was isolated from 500 l of sample using the current validated RNA isolation product (competitor), or from only 50 l of sample using the MagMAX Viral RNA Isolation Kit (Ambion). Equal volumes of the RNA recovered were used for the viral RNA detection assay. Despite using ten-fold less sample input, the MagMAX Kit produced enough RNA to allow equal or better detection of viral RNA in 9 of the 12 samples.
Figure 1. MagMAX Achieves Better Sensitivity with 10X Less Sample than Competitors Kit. The sensitivity of viral RNA detection using Ambions MagMax Kit was compared with that of the current NVSL-validated viral testing protocol (competitor). Clinical tracheal and cloacal swab samples were collected from poultry infected with California exotic newcastle disease (END) virus . RNA was isolated from 50 l of sample using the MagMAX protocol or from 500 l of sample using a competitor's product. Even with 10-fold less sample, viral RNA detection in RNA isolated with the MagMAX protocol was as sensitive or more sensitive than from RNA isolated using the competitors kit.
Advantages of Magnetic Beads for Viral RNA Isolation
Magnetic bead-based technology is extremely effective for viral RNA isolation from cell-free samples. The MagMAX technology results in better RNA capture and higher RNA yield than glass filter based techniques. Furthermore, RNA yields are also more consistent, both from experiment to experiment and over a broad range of sample sizeseven from high volume and/or low viral titer samples. Another important benefit of magnetic bead-based RNA capture over glass filter-based methods is that the problem of filter clogging with samples such as plasma and milk is eliminated. Furthermore, since only a small volume of magnetic beads is needed, bound RNA can be eluted in just 20 l of nuclease-free water or low salt buffer. This makes the kit extremely useful for concentrating viral RNA from large, dilute samples.
A Fast and Simple Processing Method
In the MagMAX procedure, plasma, serum, swab samples, or cell free samples undergo a lysis/binding step to denature proteins in the sample and to simultaneously bind the RNA on magnetic beads. The beads are then captured and washed several times, and the RNA is eluted in 20 l of nuclease-free water or low salt buffer. The procedure does not include time-consuming and difficult-to-automate protease digestion, organic extraction, centrifugation, or vacuum filtration steps. The RNA obtained with the kit can be used directly for qRT-PCR and RNA amplification, Northern analysis, and other applications.
Recover a Broad Size Range of RNA from Diverse Samples
To demonstrate the ability of the MagMAX Kit to recover a broad range of differently sized RNAs from diverse samples, lysis solution was added to samples (using water as a positive control). A mixture of eight RNA transcripts ranging in size from 100 nt to 9000 nt were spiked into the mixture. The RNA transcripts were isolated from the samples using the MagMAX Viral RNA Isolation Kit and run on an agarose gel (Figure 2). The MagMAX Kit recovered each of the eight RNA transcripts largely intacteven from raw milk and serum, which have a notoriously high nuclease content.
Figure 2. Efficient Recovery of RNA from Various Sample Types. To test recovery of RNAs ranging in size from 100 bases to 9 kb, MagMAX Lysis Buffer and 250 ng of RNA transcripts of the indicated sizes were added. The samples were then processed using the MagMAX Viral RNA Isolation Kit. The RNA obtained was analyzed on a 1% denaturing agarose gel stained with SYBR Gold. High quality RNA was effectively recovered over a large size range from common sample sources. BHI: brain heart infusion broth. VTM: viral transport media.
Linear Recovery Down to 50 Copies of Transcript
With the MagMAX Viral RNA Isolation Kit, viral RNA can be quantitatively recovered from biological fluids spiked with RNA input levels ranging over 5 orders of magnitude. This was demonstrated by spiking plasma, serum, milk, and water samples with dilutions of a 466 nt HIV Armored RNA transcriptan RNase resistant, viral protein-coated RNA molecule. A titration from 50 to 4,000,000 copies of Armored RNA was added to duplicate samples for amplification. The RNA was then recovered from the sample using the MagMAX Kit, and 20% of the sample was used in qRT-PCR to detect the HIV RNA. Figure 3 shows that RNA recovery was linear over a broad range of viral concentrations, i.e. for both low and high viral titers, including samples containing as few as 50 input copies of transcript.
Figure 3. Linear Recovery of Synthetic Viral RNA from Biological Fluids. MagMAX Lysis Buffer was added to samples of water, plasma, serum, and milk, and the mixture was then spiked with a serial dilution of a 466 nt HIV Armored RNA transcript. (Armored RNA is protected from nucleases by a protein coat derived from the MS2 coat protein gene.) Viral RNA was isolated using the MagMAX-96 Viral RNA Isolation Kit. The recovered RNA was analyzed by qRT-PCR. The results demonstrate that as few as 50 input copies of transcript can be successfully detected in RNA isolated using the MagMAX Kit.
Processing Individual Samples
The MagMAX Viral RNA Isolation Kit is ideal for proficiency testing and training applications, and for low throughput virus surveillance. It contains reagents for 50 RNA extractions from liquid samples of up to 400 l each. Samples can be processed in just 30 minutes. This kit can increase the sensitivity of diagnostic assays for low viral titer samples, since up to 400 l of sample can be used for processing. And RNA is eluted in 20 l, concentrating viral RNA 20-fold. The only equipment required is an inexpensive and durable magnetic stand.
High Throughput Processing
Magnetic bead based procedures are easy to adapt to high throughput formats, both for manual processing with a multichannel pipettor and for robotic liquid handling systems. The MagMAX-96 Viral RNA Isolation Kit is designed for high throughput viral RNA isolation of 50 l samples in 96 well plates. Using a multichannel pipettor or a robotic liquid handling system, a 96 well plate can be processed in only 30 minutes. Protocols for the MagMAX-96 Viral RNA Isolation procedure can be downloaded for the MultiPROBE II HT (Perkin Elmer) and the Biomek 2000 (Beckman Coulter) from Ambions Automation Resource Page. This version of the kit is ideal for high throughput surveillance and for screening during viral disease outbreak. Bulk quantities and custom lots are available.