NEW Maximize Viral RNA Yield from Biological Fluid

Ambion’s magnetic bead-based MagMAX™ Viral RNA Isolation Kit

Ambion’s magnetic bead-based MagMAX™ Viral RNA Isolation Kit is optimized for isolating viral RNA from biological fluids and cell-free samples such as serum, plasma, swabs, and cell culture media. RNA isolation from cell-free samples presents some unique challenges compared to RNA isolation from tissue samples or cultured cells. These include low viral titers, large volumes, and complex samples—which often contain extracellular proteins and high levels of RNase. Since researchers and diagnosticians often need to conduct large scale screening experiments for large studies or during viral outbreaks, the process must also be rapid and reproducible, as well as easily adapted for both manual and robotic high throughput processing. Finally, the procedure must be compatible with real-time qRT-PCR, the standard for diagnostic detection of viral RNA. The MagMAX Viral RNA Isolation Kit addresses these issues. It offers the advantages of solid phase extraction using glass fiber filters—it is fast and requires no organic solvents or RNA precipitation while eliminating the problems often encountered with filter-based methods, such as clogging, large elution volumes, and inconsistent RNA yield. MagMAX Kits are available for both manual and high throughput processing.

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 manufacturer’s RNA isolation product to obtain RNA from biological fluids for their validated viral testing process. When they compared this validated system to Ambion’s 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 µl of sample using the current validated RNA isolation product (competitor), or from only 50 µl 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 Competitor’s Kit. The sensitivity of viral RNA detection using Ambion’s 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 µl of sample using the MagMAX protocol or from 500 µl 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 competitor’s 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 sizes—even 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 µl 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 µl 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 intact—even 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® transcript—an 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 µl 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 µl of sample can be used for processing. And RNA is eluted in 20 µl, 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 µl 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 Ambion’s 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.

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Customizing Viral RNA Isolation for Your Research Needs An off-the-shelf product doesn’t always fit everyone’s application or sample size. Ambion scientists will work with you to tailor or optimize any of these kits to satisfy your needs.

Why Use Magnetic Beads? The same advantages that magnetic beads provide for viral RNA isolation also apply to high throughput total RNA isolation from cultured cells. No organic extraction or precipitation steps are required, there are no filters to clog, RNA is eluted in a very small volume, and the reaction is scalable. Likewise, because magnetic bead based systems do not require centrifugation or vacuum filtration steps for 96 well format processing, they are more suitable for robotic liquid handling systems than other total RNA isolation methods.

Ambion’s MagMAX™-96 Total RNA Isolation Kit is optimized for high throughput isolation of RNA from 100–105 cells and small tissue samples. Samples are lysed in a high concentration guanidinium solution. Cell debris and proteins are removed with the supernatant after pelleting magnetic beads with bound nucleic acids. DNase I is added to the lysate to digest genomic DNA. Next, the RNA is rebound to the magnetic beads and degraded DNA and DNase I are removed with the supernatant. Total RNA is eluted with either Elution Buffer or Nuclease-Free Water after two washes. Total RNA isolated with the MagMAX-96 Total RNA Isolation Kit is ready for all common molecular biology applications including RT-PCR, RNA amplification, and Northern blotting. Typically, the RNA obtained from small tissue samples using the MagMAX-96 Total RNA Isolation Kit can be used with great success in a qRT-PCR and RPA, but Ambion does not recommend its use in array analysis. The kit includes 96 well plates and reagents for 4 x 96 total RNA isolations.