exoRNeasy Midi and Maxi Kits

• Highly pure total RNA (including the small RNA fraction) from exosomes and other extracellular vesicles (EVs)
• RNA isolation exclusively from exosomes, excluding RNA from non-vesicular protein complexes
• Convenient on-column procedure provides access to RNA in just one hour
• High sensitivity provided by maxi kit that allows large sample input volumes to detect low-abundance RNAs
• Isolation from most biofluids, including urine, serum, plasma, CSF and cell culture supernatant

exoRNeasy Kits use membrane affinity spin columns to efficiently capture exosomes and other EVs from most biofluids, including urine, serum, plasma, CSF and cell culture supernatant. The kits then use proven RNeasy MinElute spin columns to isolate RNA from the EVs. The midi column format enables efficient processing of smaller sample volumes (up to 1 ml serum/plasma or 4 ml of urine), while the maxi column format allows the use of large sample volumes, up to 4 ml serum/plasma, 16 ml urine or 32 ml cell culture supernatant, to detect low-abundance RNAs with high confidence. The results are fast, consistent and highly suited to sensitive downstream applications. The RNA isolation part of the protocol using exoRNeasy Kits can be automated on the QIAcube Connect.

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The protocol for exoRNeasy purification of exosomes and other extracellular vesicles is not only faster than ultracentrifugation, but also yields a cleaner preparation. Scanning electron microscopy shows that while both techniques deliver intact vesicles of the expected size, preparation from ultracentrifugation also contains many smaller structures that do not match the expected size or shape for EVs (see figure  Intact vesicles are eluted from the exoEasy membrane with higher purity compared with ultracentrifugation).

Moreover, both exoRNeasy and the traditional ultracentrifugation method deliver similar sizes and yields of large and small RNAs, as measured by Bioanalyzer analysis (see figure  exoRNeasy isolates small and large RNAs from extracellular vesicles in plasma).

Finally, exoRNeasy captures all cell-free mRNAs in plasma, as well as EV-associated miRNAs, as confirmed by RT-qPCR experiments on the RNAs purified with the exoRNeasy protocol and the flow-through from the exoEasy column (see figure  exoRNeasy captures all mRNA and vesicle-specific miRNAs in plasma).

exoRNeasy Kits improve upon traditional ultracentrifugation microvesicle isolation methods, yielding purified extracellular vesicles in just 20 minutes. Using technology developed with Exosome Diagnostics, Inc., the kits use a spin column format and specialized buffers to capture exosomes from prefiltered biofluids; up to 32 ml using the maxi kit, and 4 ml using the midi kit. Total RNA is then extracted using QIAGEN miRNeasy technology. The entire procedure, from sample to RNA, takes only one hour.

The exoRNeasy Serum/Plasma Starter Kit provides both midi and maxi exoEasy columns, enabling analysis at varied volumes of prefiltered biofluids.

The total procedure for isolating RNA from extracellular vesicles comprises two phases: exosome purification and RNA isolation (see figure  The exoRNeasy Serum/Plasma Maxi Kit workflow — sample to microvesicles to total RNA in just 1 hour).

In the exosome purification stage, prefiltered sample (with particles larger than 0.8 μm excluded) is mixed with Buffer XBP and bound to an exoEasy membrane affinity spin column. The bound exosomes are washed with Buffer XWP, and then lysed with QIAzol.

In the RNA extraction step, chloroform is added to the QIAzol eluate, and the aqueous phase is recovered and mixed with ethanol. Total RNA, including miRNA, binds to the spin column, where it is washed three times and eluted.

exoRNeasy Kits are highly suited for isolating total RNA, including mRNA and miRNA, from microvesicles found in biofluid samples.

Applications include:

1. Transcriptomics
2. miRNA profiling
3. mRNA profiling
4. RNA sequencing