Figure 1: Monarch Mag Cell-free RNA (cfRNA) Extraction Kit enables extraction of high-quality cfRNA.

A) 2 ml plasma from six healthy donors was subjected to cfRNA extraction using the Monarch Mag Cell-free RNA (cfRNA) Extraction Kit and quantified using RNA Pico Bioanalyzer® (Agilent® Technologies). Inherent donor variability is captured in cfRNA quantification. (B) 1 ml and 2 ml plasma from pooled healthy donor set was subjected to extraction using the Monarch Mag Cell-free RNA (cfRNA) Extraction Kit and a cfRNA extraction workflow from another supplier. Resulting cfRNA concentrations are reported as measured by RNA Pico Bioanalyzer (Agilent Technologies). (C) 1 ml plasma from a healthy donor was subjected to extraction using the Monarch Mag Cell-free RNA (cfRNA) Extraction Kit and a cfRNA extraction kit from another supplier. miRNA was measured using the Small RNA Bioanalyzer (Agilent Technologies).

Figure 2: Monarch Mag Cell-free RNA (cfRNA) Extraction Kit includes an on-bead DNase treatment for effective removal of contaminating DNA.

Optimized on-bead DNase treatment removes contaminating DNA during extraction. To demonstrate the effectiveness of the methodology, sterile cell culture media (DMEM, Thermo Fisher Scientific®) was spiked with Low Molecular Weight DNA Ladder (NEB #N3233), and ssRNA ladder (NEB #N0362) to simulate biofluid samples containing DNA and RNA. 2 ml of the simulated sample was used as the input for extraction using the Monarch Mag Cell-free RNA (cfRNA) Extraction Kit, with and without incorporating the on-bead DNase step during extraction. Isolated nucleic acids were analyzed on the Cell-Free DNA TapeStation® (Agilent Technologies) for DNA visualization and High Sensitivity RNA TapeStation (Agilent Technologies) for RNA visualization. Samples treated with on-bead DNase show no trace of DNA on the Cell-free DNA TapeStation, and show successful isolation of spiked RNA ladder as seen on the HS RNA TapeStation.

Figure 3: Monarch Mag Cell-free RNA (cfRNA) Extraction Kit eluates show minimal DNA contamination compared to another supplier.

LC-MS based analysis of the composition of the cfRNA extraction eluates demonstrates minimal DNA contamination when using Monarch Mag Cell-free RNA (cfRNA) Extraction Kit. 2 ml plasma from healthy donors was subjected to cfRNA extraction using the Monarch Mag Cell-free RNA (cfRNA) Extraction Kit and a cfRNA extraction kit from another supplier. Contaminating DNA in cfRNA eluates was measured using nucleoside LC-MS, Nucleoside Digestion Mix. Monarch Mag Cell-free RNA (cfRNA) Extraction Kit results in minimal DNA contamination in the eluate, leading to true RNA representation in downstream assays.

Figure 4: Monarch Mag Cell-free RNA (cfRNA) Extraction Kit combined with NEBNext UltraExpress RNA Library Prep Kit enables high-quality cfRNA sequencing workflow.

Circulating cell-free RNA from three donors was isolated using the Monarch Mag Cell-free RNA (cfRNA) Extraction Kit. 2 μl of each extracted cfRNA was used as input for the NEBNext UltraExpress® RNA Library Prep Kit and NEBNext Multiplex Oligos for Illumina with modified protocol to prepare total RNA-seq libraries, which were then pooled into 8-plex hybrid capture reactions using an RNA exome panel to generate exome-enriched RNA-seq libraries. Both total RNA-seq and exome-enriched RNA-seq libraries were sequenced on the NovaSeq 6000 2x100 bases, downsampled to 30 million read pairs. To generate RNA-seq metrics, reads were adapter trimmed using Flexbar, aligned to the GRCh38 reference genome with STAR and deduplicated using Picard MarkDuplicates. The percentage of ribosomal RNA reads was calculated using BBDuk. Salmon was used to map to GENCODE annotations and quantify mRNA transcripts from trimmed, unique, deduplicated reads.

Figure 5: Monarch Mag Cell-free RNA (cfRNA) Extraction Kit combined with NEBNext Low-bias Small RNA Library Prep Kit enables small RNA profiling.

Circulating cell-free RNA from three donors was isolated using the Monarch Mag Cell-free RNA (cfRNA) Extraction Kit. 1 or 0.5 μl of RNA was used as input into the NEBNext Low-bias Small RNA Library Prep Kit with NEBNext LV Unique Dual Index Primers with modifications to account for the very low inputs to prepare small RNA-seq libraries. Libraries were sequenced on the NextSeq 550 1x56 bases and downsampled to 10 million total reads. Reads were adaptor trimmed using Flexbar, mapped to the GRCh38 reference genome using STAR, and STAR was used for transcript assignment and counting.

Figure 6: Monarch Mag Cell-free RNA (cfRNA) Extraction Kit workflow.

The Monarch Mag Cell-free RNA (cfRNA) Extraction Kit is designed for efficient and reproducible isolation of circulating cell-free RNA (cfRNA) from biofluids with an on-bead DNase treatment included during extraction. Flexible by design, the kit supports both manual and automated workflows.