Is the Magnazol™ RNA extraction reagent phenol-free?
No. The Magnazol™ RNA extraction reagent contains guanidinium, a powerful chaotropic agent effective for rapidly inactivating nucleases, and phenol, an organic solvent used to denature and separate proteins and DNA from RNA.
Does the Magnazol™ RNA extraction reagent recover RNA from membrane-bound vesicles in plasma?
Yes, the reagent is effective for recovering cfRNA associated with proteins and with membrane-bound particles such as platelets and exosomes.
What kind of blood collection tubes are recommended for cfRNA?
EDTA tubes are generally suitable, provided the blood is fractionated within a few hours of collection. Specialized tubes for cfRNA stabilization are sold by some vendors; however those have not been extensively evaluated with this kit..
Are the magnetic beads used in the Magnazol™ RNA extraction reagent the same as those used in the NextPrepMag™ cfDNA Isolation kit?
No, the magnetic beads are different.
Can the Magnazol™ RNA extraction reagent be used to extract RNA from platelets?
Yes. The Magnazol™ RNA extraction kit has been used to extract RNA from pelleted platelets.
How do cfRNA yields compare between platelet-rich plasma, platelet-poor plasma, and serum?
In our experience, yields of cfRNA are generally much higher for platelet-rich plasma compared to platelet-poor plasma and serum. Yields of cfRNA from serum are generally higher than from platelet-poor plasma. Data showing relative yields from these three biofluids is presented in the poster Comparison of Circulating RNA Yield and Diversity.
Is there flexibility in the volume of Elution Solution used?
The volume of elution solution can be increased, but it is not recommended to decrease the volume beyond the recommended 16 µL per 0.6 mL plasma.
Can the cfRNA be eluted in water instead of the included Elution Solution?
How should the cfRNA be stored?
For convenience, the extracted cfRNA can be stored at -20°C without degradation. For long-term storage, -80°C may be preferable, but studies have not been performed that address the benefits of storage at a lower temperature.
What is the maximum lag time between collecting the blood and centrifuging it to obtain plasma or serum?
For blood collected in EDTA tubes, fractionation should be carried out as soon as practical, ideally within a few hours. For blood collection tubes marketed for preservation of cfRNA, follow vendor guidelines. Excessive lag time between blood collection and fractionation can lead to increases in RNA released from leukocytes.
What is the recommended protocol for fractionating whole blood to obtain plasma for cfRNA isolation?
For platelet-poor plasma, an initial low-speed centrifugation (e.g. 300 x g for 10 minutes) is recommended followed by a second centrifugation of the removed plasma in a conical tube at a higher speed (e.g. 3,000 x g for 15 minutes). After the first spin, carefully remove the plasma (leave some behind over the buffy coat to avoid removing any contaminating WBCs). After the second spin, remove the plasma to a new vessel, leaving a small amount behind to avoid removing any pelleted material. Fractionation is typically performed at room temperature. Many different protocols have been described for the centrifugation steps. For example, some protocols recommend 16,000 x g for the second spin.