Viral RNA ISOLATION (Saliva, Viral transport MEDIum, etc.)
CBI offers a new system for the isolation of RNA, including viral RNA, from liquid biopsies, as well as from virus transport medium (VTM). Our RNA isolation protocol is a modification of the SubXTM cfDNA kit procedure. SubX molecules' affinity to nucleic acids allows for immediate capture and preservation from degradation in bioliquid (saliva, serum, plasma, urine, VTM) without the addition of inhibitors and/or chaotropic agents. No Proteinase K digestion is required. The standard volume of liquid biopsy for DNA isolation is 2-5 ml. Scale up to 50 ml of liquid can easily be done.
We have developed a new method of RNA isolation from liquid samples based on a dual-functional proprietary substance (SubX) that binds DNA and RNA under physiological conditions (e.g., directly from biological fluids) where it is absorbed by a solid phase Binding Matrix, e.g. magnetic beads.
Our RNA isolation procedure includes the following steps:
• RNA binding: simply add SubX and Binding Matrix to your bioliquid sample and rotate for a few minutes;
• Washing: 3 brief washing steps;
• Elution: RNA is easily eluted in a small volume (30-50µl) of the buffer.
Figure 1. Amplification curves obtained for viral RNA isolated from serially diluted saliva samples spiked with lentivirus particles (Left panel). Plotting Ct values against viral RNA copy numbers (Right panel).
Table 1. Estimation of the RNA yield from saliva sample stored at ambient conditions for 72 hours.
Hours after spiking
Ct Value (±SD)
15.03 ± 0.07
Calculated from the Lentivirus Titer
15.25 ± 0.34
2x107 ± 1.3x106
Interpolated from the Ct Plot
Estimation of the virus RNA yield from the saliva sample stored for 3 days at room temperature confirms that the RNA-SubX complex is stable under ambient conditions and preserves nucleic acids from degradation. Using Ct-plot (Fig.1) as a standard curve for interpolation of RNA copy numbers in the stored saliva sample resulted in an estimation of the 100% recovery rate. Standard error is about 6% (1.3x106/2x107=0.06).
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