FOR ISOLATION OF CIRCULATING RNA FROM PLASMA FOR SMALL RNA-SEQ
NextPrep™ Magnazol™ cfRNA Isolation Kit
- Ideal for extracting cfRNA from human plasma or serum for small RNA-seq
- Magnetic beads for rapid purification of RNA from aqueous phase
- Allows highest volume of biofluids to be processed in a single tube
- Fast, < 1 hour protocol with minimal hands-on time
- Generates quality NGS libraries using few PCR cycles
- No loss of pellet during IPA precipitation
- Kit includes BCP, an alternative to chloroform
- All steps carried out at room temperature
For research use only. Not for use in diagnostic procedures.
Extracting cfRNA from Human Plasma or Serum
The NextPrep™ Magnazol™ cfRNA isolation kit is designed for extracting cell-free RNA (cfRNA) for constructing NGS libraries from human blood plasma or serum, using a fast magnetic bead-based format requiring minimal hands-on time. The isolated cfRNA is ideal for next generation sequencing. Suitability of the RNA for use in NGS library construction has been verified using the NEXTFLEX® small RNA-Seq v3 library prep kit for Illumina® sequencing.
Cell Free RNA Isolation Challenges
Extraction of cfRNA from plasma is challenging because it is highly heterogeneous in size and present in low concentrations. Since the expected yields are low, the RNA recovered cannot be reliably detected using standard methods such as NanoDrop spectrophotometer, gel or capillary electrophoresis, or RNA binding dyes. The NextPrep™ Magnazol™ cfRNA isolation kit allows for the highest volume of biofluids to be processed in a single microfuge tube compared to other kits on market.
The NextPrep™ Magnazol™ cfRNA isolation kit delivers robust cfRNA yields and quality NGS libraries with a high percentage of reads that map to miRbase than other kits on market and allows for miRNA diversity exploration using the NEXTFLEX® small RNA-Seq v3 kit.
NextPrep™ Magnazol™ cfRNA isolation kit outperforms the Qiagen® miRNeasy serum/plasma kits and Thermo Fisher® Scientific Trizol™-LS reagents.
Cell Free RNA Isolation Protocol
This cell free RNA isolation procedure, which can be completed in less than 1 hour, includes an initial lysis/RNase inactivation, followed by phase separation, magnetic bead-based RNA binding step, wash steps, and elution of cfRNA from the magnetic beads.
PLASMA/SERUM + EXTRACTION REAGENT + BPC (5 MIN)
AQUEOUS PHASE + MAGNETIC BEADS (10 MIN)
ATTRACT TO MAGNET AND REMOVE FLUID
ELUTE (10 MIN)
Small RNA-seq data generated using the NEXTFLEX® small RNA-Seq v3 kit to assess miRNA mapped reads using cfRNA extracted from platelet-rich plasma using various extraction methods
- Magnazol™ Extraction Reagent
- Magnazol™ Magnetic Beads
- BCP (1-bromo-3-chloropropane)
- RNA Elution Solution (0.1 mM EDTA)
REQUIRED MATERIALS NOT PROVIDED
- Ethanol, 100% and 80%
- Vortex mixer
- Platform shaker, tube rotator, or heat block
- Microcentrifuge or “picofuge”
- 2 mL microfuge tubes, nuclease-free. Smaller tubes may be used for the elution step.
- Pipettors (P-1000, P-200, P-20 or equivalent) and tips
- Magnetic stand for microfuge tubes
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.
- Comparison of Circulating RNA Yield & Diversity in Platelet-Poor Plasma, Platelet-Rich Plasma, & Serum from Healthy Individuals
- Complete, streamlined, reduced-bias workflow for RNA extraction and small RNA library preparation from serum and plasma samples
The NextPrep™ Magnazol™ cfRNA isolation kit contains sufficient reagents for extraction of RNA from 25 samples of up to 0.6 mL plasma. The kit components are stable for at least 12 months from time of receipt, when properly stored.