RNA isolation is the process of extracting ribonucleic acid (RNA) molecules from a biological sample. RNA is a nucleic acid that plays a key role in many cellular processes, including the synthesis of proteins. In recent years, RNA isolation has become increasingly important in various fields of biology and medicine, including genomics, therapy and diagnostics.
One area where RNA isolation is particularly useful is the study of exosomes. Exosomes are small, membrane-bound vesicles that are released by cells into the extracellular environment. They play a critical role in intercellular communication and have been implicated in a wide range of physiological and pathological processes.
When it comes to isolating exosomes from serum or plasma for later RNA analysis, there are several methods that can be used. These methods vary in terms of their efficiency, sensitivity, and specificity, and the best approach will depend on the specific goals of the experiment and the characteristics of the sample.
▌RNA isolation from exosomes by centrifugation
One commonly used method for exosome isolation from serum or plasma samples is ultracentrifugation. This method involves spinning the sample at high speed in a special centrifuge, which allow the exosomes to be separated from other components of the sample based on their size and density. This method is relatively efficient and can be used to isolate large amounts of exosomes. However, it is not always very specific, and can result in the co-isolation of other types of vesicles or contaminants.
Subsequently, to isolate RNA from exosomes, researchers first collect the biological sample containing the exosomes. The sample is then treated with a reagent, such as a detergent or phenol–chloroform, to lysis the exosomes and release their contents. Specifically we recommend the use of guanidine isothiocyanate for exosome lysis because also prevents the RNase and DNase enzymes from acting and denaturing DNA and RNA.
Next, the sample is subjected to ultracentrifugation, a process in which it is spun at high speeds in a special centrifuge. This causes the exosomes and other large particles in the sample to sediment to the bottom of the centrifuge tube, while the smaller molecules, including RNA, remain in the supernatant.
The RNA-containing supernatant can then be collected and purified using a variety of techniques, such as column chromatography or a spin filter. This purified RNA can then be used for downstream applications, such as reverse transcription and PCR, to study the exosomes and their role in the sample.
▌RNA isolation from exosomes by chromatography
Another method that is often used for exosome isolation from serum or plasma samples is size-exclusion chromatography (SEC). This method involves passing the sample through a special column that is designed to separate molecules based on their size. Exosomes, which are typically 30-100 nanometers in diameter, can be efficiently isolated using this method. However, this method can be time-consuming and may not be suitable for all types of samples.
To purify RNA from exosomes isolated using size-exclusion chromatography, researchers first collect the biological sample containing the exosomes. The sample is then treated with phenol–chloroform or detergent protocol, to lysis the exosomes and release their contents. As we mention before the use of guanidine thiocyanate reagents based it is recommend, for RNase inactivation as they can rapidly degrade RNA.
Next, the sample is passed through a size-exclusion chromatography column, which is packed with beads or gel with pores of different sizes. As the sample flows through the column, the exosomes and other large particles in the sample are excluded from the pores and are collected in the void volume, while the smaller molecules, including RNA, pass through the pores and are collected in the eluted fraction.
Nevertheless, we recommend the RNA purification from exosome isolated from serum or plasma by SEC and later lysed with phenol_choroform protocol by slica adsorption, using spin column or beads.
Overall, size-exclusion chromatography is a powerful tool for isolating exosome from other small particles in biological samples and it is perfectly compatible with the most common based methods for the extraction and subsequent analysis of RNA, which it is widely used in research and has provided many insights into the role of exosomes in health and disease
Immunostep offers some of the most common techniques for exosomes isolation with all the guarantees. Below we describe some of the main advantages of Immunostep Exosome isolation columns kit (Size-exclusion chromatography):
► Great performance separating exosomes from important contaminants: Immunostep reusable Size Exclusion Columns allows the rapid isolation of extracellular vesicles (EVs) from cell culture supernatants and complex biological fluids. Each column efficiently removes background proteins, lipids, solutes, and other contaminants to improve the sensitivity and accuracy of downstream analysis while maintaining the biological properties of the EVs.
► Fast and efficient exosome isolation kit: The vesicles can be isolated in 15 minutes thanks to these columns, and this allows EV samples to be collected and analyzed very fast. These Columns are ready to use, and quality assured so your isolations are reproducible from column to column and batch to batch.
► Reusable exosome isolation columns and efficient packaging: Immunostep columns have been developed to facilitate their use, and for this reason it comes with a “smart box” which easily transforms to a practical column rack.
► Improved exosome isolation and recovery: A comparison was made using columns competitor, obtaining Immunostep columns a better performance in terms of recovery (discover more about exosome isolation columns performance).
▌RNA isolation from exosomes by immunoaffinity purification
A more recent method that has gained popularity for exoxome isolation from serum or plasma samples is immunoaffinity purification. This method involves using antibodies that specifically recognize and bind to proteins on the surface of exosomes. The antibodies are attached to a solid support, such as beads or a column, and the sample is passed over the support. The exosomes in the sample will then bind to the antibodies and be retained on the solid support, while the other molecules in the sample will flow through. The exosome-bound fraction can then be collected and washed to remove any non-specifically bound molecules.
The exosomes can then be isolated using magnetic beads or other techniques that allow the bound exosomes to be separated from the rest of the sample. This method is highly specific and can be used to isolate pure populations of exosomes.
To isolate RNA from exosomes using immunoaffinity purification, researchers first collect the biological sample containing the exosomes. The sample is then treated with a reagent, as we mention before, to lysis the exosomes and release their contents.
And as for other methods, the RNA can then be released from the exosomes by treating the fraction with a chaotropic agent, such as guanidine isothiocyanate, and purified using a variety of techniques, such as column chromatography or a spin filter.
In this sense, at immunostep we have developed ExoStepTM kit which is an immunobead assay for the detection of exosomes using a bead-bound capture antibody and fluorochrome conjugated detection antibody. This kit provides reproducible results and is intended for flow cytometry analysis of pre-enriched human exosomes from biofluids (plasma, serum, urine, among others) or cell culture media. It should be noted that the components and method used in this kit (ExoStep) do not interfere with the usual RNA purification protocols that we have discussed here.
▌The best RNA isolation method from exosomes
We conclude that there is no «best» method for isolating RNA from exosomes of serum samples, as different methods may be more or less suitable depending on the specific research question and the characteristics of the sample being studied.
But, as we have previously approached some commonly used methods for exosome isolating for downstream RNA purification and analysis include ultracentrifugation, size-exclusion chromatography, and immunoaffinity purification.
Ultimately, the best method for RNA isolation from exosomes will depend on the specific goals of the experiment and the characteristics of the sample. In general, it is important to choose a method that is efficient, sensitive, and specific, and that allows the isolation of high-quality RNA from exosomes. In some cases, it may be necessary to combine multiple methods in order to achieve the best results.
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