Important Procedure Notes when using the Monarch^® Mag Cell-free RNA (cfRNA) Extraction Kit (NEB #T4080)

Cell-free RNA characteristics

Cell-free RNAs (cfRNAs) serve as important biomarkers in liquid biopsy for the detection and monitoring of cancer and other diseases. Cell-free RNAs are fragmented and can exist in diverse fragment sizes ranging from small microRNAs to long non-coding RNAs. These RNAs are free-circulating in body fluids or associated with extracellular vesicles (EVs). All forms of cfRNAs are present in extremely low concentrations and variable amounts depending on physiological conditions and disease states. Key preanalytical conditions in cfRNA testing workflows include cfRNA preservation at harvest, a sensitive extraction process that enables a faithful representation of cfRNA sizes, and removal of contaminating DNA that can lead to false positives in downstream RNA-based workflows.

Sample collection

Sample collection and preservation play a crucial role in cell-free RNA yield and recovery. Sample collection tubes and collection processes should be chosen carefully to stabilize cell-free nucleic acids and minimize contamination by cellular nucleic acids. Tube manufacturer’s instructions for sample collection and storage should be followed. This kit is compatible with several different collection tubes including standard anticoagulant tubes such as EDTA and sodium citrate, as well as preservative-containing tubes such as those offered by Streck, Norgen and PAXgene. Heparin containing blood collection tubes are not recommended due to downstream interference of heparin in molecular assays.

Cell-free RNA size range

The standard protocol of this kit enables efficient extraction of all sizes of cfRNA, as verified by total RNA-Seq. Protocol modifications are provided on the product webpage for modulating the RNA fragment sizes recovered.

Sample types

This extraction kit has been evaluated for plasma, urine and cerebrospinal fluid (CSF) samples. 

Storing and handling of Monarch Mag Beads M2

Store Monarch Mag Beads M2 at 4°C after first use. For optimal performance, Monarch Mag Beads M2 should be equilibrated to room temperature before use. Importantly, the beads should be thoroughly vortexed right before dispensing into the sample.

Binding of cfRNA to beads

RNA binds to silica-coated magnetic beads through interactions with the bead surface in the presence of the binding buffer (Monarch Buffer BX) and the appropriate volume of isopropanol. At this stage, the sample-binding mix needs to be mixed for 10 minutes to ensure efficient binding of cfRNA. A thermal mixer, shaker or an end-over-end mixer is recommended.

On-bead DNase treatment

Monarch DNase I, Lyophilized supplied with the kit contains the optimal enzyme units needed for complete DNA removal during cfRNA extraction from biofluid samples. This DNase I is a RNase-free formulation. Rehydration of supplied lyophilized DNase must be performed using the instructions in this manual as the final enzyme units required for cfRNA extraction are different from other kits and applications. After the DNase reaction mix is added to the sample, a 15 min incubation with intermittent mixing is recommended. During RNA rebinding with Monarch StabiLyse DNA/RNA Buffer and isopropanol, it is important to allow 5 mins of binding, with shaking, to ensure RNA is rebound to the beads, while the digested DNA remains in the supernatant.

Bead drying

Following the final wash, beads should be dried thoroughly to remove residual ethanol. Optimally dried beads appear shiny and uniformly dark, while over-dried beads appear cracked or matte light brown. Avoid over-drying the beads by following the recommended drying times.

Elution volume considerations

At the elution step, RNA is released from the magnetic beads using nuclease-free water. For optimal release of all bound cfRNA, an elution volume of 100 µl is recommended, followed by a magnetic bead-based concentration into smaller volumes. Depending on the workflow design and volume requirement for the downstream assay, some users may prefer to elute in <100 µl. Vigorous mixing on a thermal mixer or a vortexer is recommended to ensure complete release of RNA into the water. For automated workflows, adjust minimum elution volumes according to the specifications and requirements of the system being used.

Concentrating cfRNA 

Because of the intrinsically low concentrations of cell-free RNA, the extracted cfRNA may need to be concentrated into smaller volumes before use in downstream applications. The beads and buffers included in the kit are sufficient for concentrating cfRNA using the concentration protocol provided in the manual. Other options for concentrating cfRNA include column-based kit such as the Monarch Spin RNA Cleanup Kit (10 µg) (NEB #T2030) or vacuum concentrators such as SpeedVac.

Cell-free RNA Quantitation and Downstream Analysis

Due to the low concentration of cell-free RNA circulating in biofluids, UV-vis spectrophotometric methods are not recommended for quantification. Additionally, fluorometric methods such as Qubit™ (Invitrogen) may not provide accurate quantification since the dye is unsuitable for the conformation/length of cfRNA as per the Qubit manufacturer. Sensitive automated electrophoresis such as Agilent RNA 6000 Pico Bioanalyzer is strongly recommended. Quantitative RT-PCR using primer/probes that specifically only target RNA signal and not DNA are also gives a more accurate assessment of cfRNA concentrations. 

The extracted cell-free RNA is application-ready and can be used in sequencing or amplification-based detection. For sequencing-based analysis, NEBNext library prep solutions are recommended.