EVeryRNA™ EV RNA Purification System with SmartSEC Single (10 reactions)

To understand how well SmartSEC Single performs compared to a competitor’s q SEC columns, we isolated EVs from 250 µL of human serum using both SmartSEC Single and q SEC columns and analyzed 1 µg of protein equivalent from each isolation method using fluorescent nanoparticle tracking analysis (fNTA, Figure 3). EVs isolated using SmartSEC Single show a higher number of particles per mg of protein (Figure 3) than EVs isolated using the q SEC columns.

EVeryRNA captures EVerything

To demonstrate the ability of the EVeryRNA EV RNA Purification System to capture the full range of RNAs, we used the kit to isolate RNA from 10,000 cells (Figure 4, lane 1), from EVs that were isolated from 250 µL of serum using SmartSEC Single (Figure 4, lane 2), and from buffer spiked with 0.1 pmol of Cel-miR-39 (Figure 4, lane 3). The high quality of the isolated RNA can be seen in lane 1, where the RNA integrity number (RIN) is 9.9 and the 28S/18S RNA ratio is 1.5.  The multiple bands in lane 2 demonstrate that EVeryRNA captures RNAs of different lengths—EVerything—from EVs with no apparent bias or size preference. The strong signal from the spiked-in miRNA in lane 3 demonstrates the good recovery of even small RNAs.

EVeryRNA works well with SmartSEC Single, yielding broad and unbiased size distribution of RNAs (Figure 5).

EVeryRNA delivers similar amounts of RNA as phenol-based methods

To demonstrate the excellent RNA yields obtained with EVeryRNA, we isolated EVs from 250 µL of serum using SmartSEC Single, spiked in 0.1 pmol of Cel-miR-39, and used both EVeryRNA and a phenol-based kit to isolate RNA. The isolated RNA was reverse transcribed using the EVeryRNA cDNA Synthesis & Pre-amplification Kit and the copy number of Cel-miR-39 measured (Figure 6). The EVeryRNA EV RNA Purification System delivered similar levels of Cel-miR-39 as the phenol-based method.

EVeryRNA efficiently isolates mRNA

We used the EVeryRNA EV RNA isolation kit to isolate mRNA from cells overexpressing eGFP (Figure 7). Robust levels of eGFP mRNA are recovered when cells are overexpressing eGFP.

miRNA isolated using EVeryRNA can be used for miRNA profiling and RNA-seq

We isolated EVs from 250 µL of serum using SmartSEC Single and used the EVeryRNA EV RNA Purification System and EVeryRNA cDNA Synthesis & Pre-amplification System to isolate and reverse transcribe EV RNAs for miRNA profiling using the SeraMir Human Exosome RNA Profiling Plate. We were able to detect a number of miRNAs both with amplification (96 miRNAs) and without amplification (16 miRNAs, with 14 overlapping with the miRNAs detected with amplification, Figure 8).

We were also able to show robust, successful RNA-seq runs using RNA isolated from EVs with EVeryRNA (Table 2). All three EV isolation methods tested generated high-quality RNA-seq data.

Table 2. Successful RNA-seq with EVeryRNA-isolated EV RNA