EVeryRNA™ EV RNA Purification System with ExoQuick®-TC EV Isolation

Maximize productivity with ExoQuick and EVeryRNA

Isolate EVs with ExoQuick-TC

Isolating EVs from tissue culture media, saliva, urine, follicular fluid, breast milk, and other biofluids with ExoQuick-TC is quick and easy, with a simple workflow that requires only 10-minutes of hands-on time (Figure 1).

To isolate exosomes from tissue culture media, simply:

• Add an appropriate volume of ExoQuick-TC
• Incubate overnight at 4°C
• Isolate exosomes with a 30-minute low-speed spin (1500g).

Isolated exosomes can be found in the pellet and resuspended in an appropriate solution.

Figure 1. The ExoQuick-TC workflow.

Purify EV RNA with EVeryRNA

The EVeryRNA EV RNA Purification System delivers high yields of highly concentrated RNA from already isolated EVs. The column-based workflow is easy to implement and can be completed in less than 30 minutes (Figure 2).

Figure 2. The quick and easy EVeryRNA EV RNA purification workflow.

Characterizing ExoQuick-TC exosomes with NanoSight

Exosomes purified with ExoQuick-TC from tissue culture media show the expected particle size distribution and high concentration yields when analyzed using NanoSight’s Nanoparticle Tracking Analysis (NTA, Figure 3).

 

Figure 3.Exosome size distribution and yields from tissue culture media. Cells from a human HT1080 lung sarcoma cell line were cultured in conditioned media (serum-free) for 72 hours. 10 mL of media was combined with 2 mL ExoQuick-TC, incubated overnight at 4°C, and centrifuged at 1500g for 30 minutes to isolate exosomes. The exosome pellet was resuspended in 1 mL PBS, diluted 1:40, and visualized on the NanoSight LM10 instrument. The analysis shows that ExoQuick-TC recovered 133 nm exosomes at a concentration of 1.74 x 10⁹ particles/mL.

 

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.

Figure 4. EVeryRNA captures EVerything.

EVeryRNA works well with ExoQuick, 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.