RNA ISOLATION

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 addition of inhibitors and/or chaotropic agents. No Proteinase K digestion is required.  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 to 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 (30-50ul) of 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)
RT-qPCR Assay

RNA copies

Notes

~1 hr

15.03 ± 0.07

2x107

Calculated from the Lentivirus Titer

72 hrs

15.25 ± 0.34

2x107 ± 1.3x106

Interpolated from the Ct Plot

Estimation of the virus RNA yield from the saliva sample stored during 3 days at room temperature confirms that RNA-SubX complex is stable at ambient conditions and preserves nucleic acids from degradation. Using Ct-plot (Fig.1) as standard curve for interpolation of RNA copy numbers in the stored saliva sample resulted in estimation of the 100% recovery rate. Standard error is about 6% (1.3x106/2x107=0.06).

 

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