RNA isolation kit viral from saliva medIum serum, plasma SubX 100 preps

RNA isolation kit, 100 preps

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.

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).

Our technology is based on using the proprietary bi-functional substance (SubX TM ) that extracts molecules of nucleic acids from tight protein complexes and binds them under physiological conditions (i.e., directly in biological liquids), followed by adsorption of the nucleic acids on a solid phase matrix, e.g. silica coated magnetic beads. Our protocol allows scaling up or down for large or extra low volumes of starting material, as well asautomation. The whole procedure can be completed within 30 min, and the purified RNA iseluted in a small volume of TE buffer.

1. SubX TM captures RNA via phosphate groups and allows for elimination of bias related to both sequence content and fragments length, thus improving extraction efficacy and accuracy of downstream applications.
2. SubX TM extracts RNA from virions and efficiently replaces (squeezes out) viral proteins. Binding to SubX TM stabilizes RNA at ambient conditions, e.g. at room temperature eliminating additional procedures for RNA purification and storage.
3. SubX TM protocol does not require additional solutions or chaotropic salts for RNA capture, therefore no dilution of starting material occurs, and the RNA isolation and concentration from the entire volume of a biological sample can be done at once.

• Simple protocol for isolation of viral RNA from any volume (0.1 ml to &>50 ml) of a biological liquid, nasopharyngeal aspirates (NPA), or nasopharyngeal swabs (NPS) collected into a viral transport medium (VTM).
• Optional isolation and separation of RNA and DNA from the same sample.
• Low cost.