Automated Workflow for High Quality, High Throughput Viral RNA

High Quality, High Throughput Viral RNA

High quality viral RNA for veterinary assays is essential for rapid viral identification. The ability to perform high throughput viral RNA isolation from diverse samples also increases laboratory efficiency, reduces labor cost, and improves viral detection. This article describes how the Ambion® MagMAX™ magnetic bead-based technology has been successfully adapted for automated sample processing on the Caliper Life Sciences Sciclone® ALH 3000 Workstation for nucleic acid isolation from diverse samples.

High throughput viral RNA isolation from diverse samples enables rapid viral detection in animal samples. The Ambion MagMAX-96 Viral RNA Isolation Kit is designed for viral RNA/DNA purification from a variety of sample types, such as animal tissues, blood, serum, plasma, milk, swabs (e.g., oral, cloacal), and feces (see sidebar, Challenges of RNA Isolation From Cell-free Systems). Small elution volumes (20–50 µL) provide concentrated, purified nucleic acids for convenient streamlining of downstream applications.

Applied Biosystems scientists with the Custom Services group (see article, Accelerate Your Research with Custom Solutions) have validated the MagMAX-96 Viral RNA Isolation Kit for use on an automated sample handling platform, the Sciclone ALH 3000 Workstation (Caliper Life Sciences). RNA was isolated from Armored RNA® particles (Asuragen, Inc.)—synthetic viral particles containing an RNA oligonucleotide—spiked into diverse biological sample matrices, including human plasma, bovine serum, raw bovine milk, and brain heart infusion (BHI) medium, which is commonly used for swab sample storage.

Recovery of RNA from Armored RNA® particles was highly consistent across the various matrix types, and no cross-contamination was observed (Figures 1–2). The data clearly indicate that the combined use of the MagMAX-96 Viral RNA Isolation Kit on the Caliper Life Sciences Sciclone ALH 3000 Workstation provides a quick and robust workflow for efficient, high throughput viral RNA/DNA isolation.

Figure 1. No Cross-Contamination was Detected When the MagMAX™-96 Viral RNA Isolation Kit was Automated on the Sciclone® ALH 3000 Workstation. Human plasma, bovine serum, raw bovine milk, and brain heart infusion (BHI) medium (50 μL) were each distributed to 3 columns of a 96-well plate. Armored XenoRNA™ particles (Asuragen, Inc.) were spiked into a subset of wells. The plate was then processed according to the MagMAX-96 Viral RNA Isolation Protocol using the Sciclone ALH 3000 Workstation (Caliper Life Sciences). XenoRNA target amplification (TaqMan® Gene Expression Assay) from the samples corresponded perfectly to the XenoRNA negative and positive sample distribution in the plate—no cross contamination was observed. The three asterisks denote no amplification in the wells that were not spiked with Armored XenoRNA particles.

Figure 2.Highly Consistent RNA Recovery with the MagMAX™-96 Viral RNA Isolation Kit When Automated on the Sciclone® ALH 3000 Workstation. To monitor consistency of recovery across a 96-well plate, lambda DNA (0.25 ng/sample) was added to the lysis/binding solution. Analysis with qPCR targeting lambda DNA and with the qRT-PCR for the XenoRNA™ amplification was performed with TaqMan® Gene Expression Assays. Amplification of lambda DNA across the plate and amplification of XenoRNA in the spike-positive wells was highly consistent with average CT values of 32.4 ± 0.25 and 28.6 ± 0.46, respectively.

Figure 3. mRNA Profiles of Mouse Tissues Stored in RNAlater. Various mouse tissues were stored in RNAlater for 1 or 4 weeks at 4°C. RNA was isolated from each tissue and analyzed using the RPA III™ Kit. 10 µg of total RNA was hybridized with 5 x 104 cpm of each of 5 combined antisense RNA probes, digested with RNase and precipitated. Products were assessed on a 5% polyacrylamide / 8 M urea gel and exposed to film for 4 hours at -80°C with an intensifying screen.

Figure 3 (Sidebar). MagMAX™-96 Viral RNA Isolation Protocol.

Challenges of RNA Isolation From Cell-free Systems

RNA isolation from cell-free samples (e.g., serum, plasma, swabs, and cell culture media) presents unique challenges compared to RNA isolation from tissues or cultured cells. These include low viral titers, large volumes to be processed, and sample complexity.

MagMAX™ magnetic bead-based technology provides a fast, simple, and easily automatable method for viral RNA and DNA isolation (Figure 3, bottom of page). DNA and RNA input levels ranging from 20 copies to 2.5 x 10⁷ copies can be quantitatively recovered from plasma, serum, and milk.

Use of MagMAX beads results in more consistent RNA recovery and more thorough DNA treatment than glass fiber filter-based RNA methods, as the RNA-binding beads can be fully resuspended in solution to enable more thorough mixing, washing, and elution.