Introduction

Traditionally, RNA is isolated by phenol/chloroform extraction, which is not amendable to high throughput methods because of the difficulty of extraction with a multi-channel pipette and the high risk of cross contamination. Solid phase binding methods have thus been developed to replace phenol/chloroform extraction for high throughput RNA isolation. Glass fiber filters and beads are among the best choices of binding matrix. While filter based viral RNA isolation is useful for single tube procedures, it may be problematic for high throughput applications because of the potential for filter clogging and the risk of cross contamination. We have developed micro-spherical bead-based RNA isolation technology. This technology has been successfully employed for Exotic Newcastle Disease diagnosis with avian swab samples by the CAHFS lab at UC Davis. A well-trained person can process 4 x 96 samples in parallel in 1.5 hours .

Performance Comparison Between Filter and Bead-Based RNA Isolation from Cell Cultures




Figure 1. RNA Quality and Yield. Total RNA was isolated from 5 x 105 K562 cells in 96-well format (Left: Filter Plate; Right: Magnetic beads) and analyzed with an RNA Chip on an Agilent 2100 bioanalyzer. The sharp shape of the ribosomal RNA peaks and the roughly 2:1 ratio of the 28S/18S indicate clean and intact RNA was obtained with both methods. The bead-based RNA isolation method typically gave higher yields of RNA.




Figure 2. RNA Linear Recovery and Consistency. Total RNA was extracted from K562 suspension cell quantities of 32, 160, 800, 4000, 20000 and 100000 cells in 96-well format (Left: Filter Plate; Right: Magnetic beads). Total RNA yield was ~ 18 pg/cell. Total RNA recovered was linearly proportional to cell input as quantified by real-time RT-PCR using a human GAPDH primer/probe set on an ABI7700 sequence detection system. RNA yield is more consistent with the bead method.

Bead-Based High Throughput Viral RNA Isolation

1. Exotic Newcastle Disease (END) Viral Isolation from Avian Swabs.

The technology has been successfully used for high throughput END diagnosis by viral RNA isolation followed by qRT-PCR during the 2002/2003 END outbreak in California. Over 100,000 swab samples were processed during the outbreak. All data in this section were generated at the CAHFS lab of UC Davis.

Figure 3. Linear Recovery of END Viral RNA and Sensitivity of Bead-Based Viral RNA Isolation Technology. An END positive sample was diluted up to 1 million-fold with viral transfer medium and subjected to bead-based viral RNA isolation. One-step qRT-PCR was used to detect ENDV from the isolated RNA. Very reliable detection can be achieved with as little as 1 nL of sample.




Figure 4. Reproducibility of Viral RNA Isolation Using Ambion Bead Technology. Three different technicians followed the same protocol to isolate viral RNA from 31 avian swab samples and quantify isolated RNA by real time RT-PCR. They had the same overall performance. The difference among them was <0.5 Ct for any given sample processed.




Figure 5. Example of END Viral Isolation from Avian Swab Samples Followed by qRT-PCR for END Diagnosis. Samples 3-6 were diagnosed and confirmed as END positive. Those samples that did not show any signal in qRT-PCR were all confirmed negative. There are no false positive or false negative results. No cross contamination was observed.


2. Bovine Viral Diarrhea Virus (BVDV) Viral RNA Isolation from Bovine Serum and Plasma.

We have successfully adapted the bead technology for viral RNA isolation from serum and plasma samples. When Armored RNA is used as a control, we can successfully recover the viral RNA with <1000 copies of Armored RNA input from both serum and plasma samples. For field BVDV-positive serum sample, we can successfully isolate and detect BVDV viral RNA with as little as 50 nL from both serum and plasma samples.




Figure 6. Linear Recovery of Viral RNA from Plasma and Serum Using Armored RNA as a Control. EV Armored RNA was spiked in 50 µl plasma or serum, and viral RNA was isolated following the standard bead protocol. 25% of the isolated viral RNA was used for qRT-PCR.





Figure 7. BVDV Viral RNA Isolation from Serum and Plasma Samples with the Bead Technology. A typical BVD-positive field sample was diluted with BVD-negative serum or plasma, and viral RNA was isolated from 50 µl diluted sample.

Conclusion

A magnetic bead based method has been developed and demonstrated to satisfy the critical requirements for high throughput viral RNA isolation from various sample matrixes. The characteristics of the method include:

  • magnetic bead-based method; no filter clogging
  • adaptable to single tube or 96-well plate isolation for high throughput needs
  • small elution volume (<20 µL), enabling using all isolated RNA for qRT-PCR
  • high sensitivity, low cross contamination, and consistent results
  • flexible for customization

Acknowledgement

We thank Dr. Sharon Hietala’s Lab (CAHFS), especially Dr. Beate Schikora, for their great help in amending and validating Ambion bead technology for high throughput END viral RNA isolation, and for sharing their great results. Many thanks to Dr. Kathy Kurth’s lab (WVDL) for supplying BVDV samples and qRT-PCR reagents for BVDV RNA quantification, and helpful discussion on protocol optimization.