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microRNA technology FAQ: 8 important questions

microRNA technology FAQ: 8 important questions.  What are the ingredients in exosome? Are most of the free microRNA in the exosome or outside of the exosome?

microRNA technology FAQ:

Q1: Are most of the free microRNA in the exosome or outside of the exosome?

A: Free microRNA in peripheral blood mainly exists in two forms, one is in exosome, and the other is binding to RBP (RNA binding protein) outside the vesicle. The proportions of these two types fluctuate, depending on different physiological or pathological situations. On the whole, most of the free microRNAs are outside the vesicles. A study of free microRNAs in plasma/serum of healthy people suggested that about 66%-68% of free microRNAs are outside the vesicles in the form of microRNA-RBP (such as Ago2) complexes. [Proc. Natl. Acad. Sci. (2011) 108: 5003-5008]. The results of another study on serum samples of prostate cancer patients showed that only about 2.5% of microRNAs are in the vesicles [Pro. Natl. Acad. Sci. (2014) 111:14888-14893].

Q2: What are the ingredients in exosome?

A: Exosome is an active vesicle secreted by a cell. In addition to vesicles, there are often abundant lipid molecules, carbohydrate molecules, as well as various protein, DNA and RNA molecules. The details are as follows:

In addition to a large number of proteins involved in the regulation of vesicle secretion, the proteins contained in exosomes also contain many proteins related to various signaling pathways. The protein components contained in exosome have tissue and cell specificity, and are also closely related to physiological and pathological conditions. A proteomic analysis of exosomes secreted by colon cancer cells has identified as many as 1,800 protein molecules with very complex protein composition. (As shown below)

Further studies have shown that the protein composition and content of exosomes are closely related to the expression of intracellular proteins secreting the vesicles, but they do not correspond exactly to the same amount [Mol. Cell.Proteomics (2013) 12: 343-355].

The RNA molecular components contained in exosome are also very complex, including mRNA, microRNA, ribosomal RNA, lncRNA, tRNA, etc. According to the existing results of RNA sequencing in exosome, the content of microRNA is the highest (76.20%). Most of the mRNA is in the form of degraded fragments (the overall content is <2%), and there are reports suggesting that there are some intact mRNAs, but the content is extremely low [BMCgenomics (2013) 14: 319].

The DNA contained in exosome includes both single-stranded DNA and double-stranded DNA. Some studies have used whole-genome sequencing to detect the entire genome sequence of tumor cells in exosomes secreted by melanoma cells [Cell Res. (2014) 24:766-769], and have a high degree of consistency with the tumor cells that secrete it. .

Q3: What are the methods for exosome extraction?

A: There are four main methods for exosome extraction:

Ultracentrifugation

This method is mainly based on the specific gravity of exosome, and exosome separation can be achieved by stepwise differential centrifugation or density gradient centrifugation. This method has low cost and high exosome yield. The disadvantage is that it is time-consuming and labor-intensive, and it is not suitable for a large number of samples.

Ultrafiltration (ultrafiltration) or size exclusion chromatography (sizeexclusion chromatography)

This method is mainly based on the size of the exosome (30-150nm, most of which is around 90-100nm), using a specific pore size ultrafiltration membrane or solid phase medium to achieve exosome separation. This method has high purity. The disadvantage is that the separation process is easily interfered by residual protein, and the elution and recovery of the complete exosome are difficult. Commercial kits, such as Vivaspin20 and 100kDa MWCO (GE) (ultrafiltration method), Sepharase 2B and CL-4B (chromatographic method)

Macromolecular polymer precipitation

This method is mainly based on the hydrophobic characteristics of exosomes, using macromolecular polymers to gather a large amount of exosomes together (as shown in the figure below) to form a larger volume of precipitate, so that exosomes can be separated by conventional centrifugation. The advantage of this method is that the separation operation is simple and fast, and it is convenient to realize batch processing of samples. Commercial kits, such as ExoQuick (SBI).

Immunoaffinity purification

This method is mainly based on exosome vesicles that show specific expression of some antigens (such as CD9, CD63, CD81), and the use of antibodies to these antigens for immunoaffinity purification and separation of exosomes. The advantage of this method is that the initial volume of the sample required is small, the specificity is strong, and different types of exosomes can be distinguished, but the disadvantage is that the cost is high. Commercial kits, such as Exo-Flow (SBI).

Q4: How to evaluate the extracted exosome?

For the extracted exosome, the following quality control can be carried out according to the conditions of the laboratory:

Electron microscopy: The advantage is the “gold standard”, which can visually see the morphological characteristics of vesicles, but the disadvantage is that the experiment is more complicated and requires specific equipment.

Vesicle particle size measurement: the advantage is the “gold standard”, the size distribution of the purified vesicles can be visually seen, so as to clarify the purity of the vesicles, the operation is simple, the disadvantage is that specific equipment (NanoSight, Particle Metrix, etc.) is required .

Vesicle-specific protein immunoblotting: WB was used to detect the expression of vesicle-specific protein in the extracted exosomes. The advantage is that it is simple and easy to implement, but the disadvantage is that the integrity and content of vesicles cannot be determined.

Vesicle-specific protein flow cytometry: label the extracted exosomes with specific dyes, then incubate them with microbeads coupled with vesicle-specific proteins, and finally use flow cytometry. The advantage is that it is simple and easy to perform and can analyze the integrity of the vesicles, but the disadvantage is that the cost is higher.

Q5: How to save the extracted exosome?

A: For short-term storage, the exosome can be stored at 4°C for a week; for long-term storage, the exosome can be stored at -20°C or -80°C. It is not recommended to freeze and thaw the frozen exosome multiple times, as this will destroy the integrity of the exosome.

Q6: What methods can be used to detect microRNA in exosome?

A: Basically, all conventional microRNA detection methods can be used for microRNA detection in exosomes, such as sequencing, microarray, and quantitative PCR, but some specific optimizations may be required in the operating methods.

Q7: What are the biggest challenges in detecting free microRNA in peripheral blood?

The most challenging aspects of free microRNA detection in peripheral blood are as follows:

Because the content of microRNA in plasma/serum is too low, the extraction and reversal efficiency is unstable, and there are large differences between batches.

Optimized scheme: add precise and quantitative, artificially synthesized microRNA from other species as spike-in reference, and evaluate the difference in microRNA experiments between different batches by detecting the content of this microRNA.

The composition of serum/plasma is complex and contains some substances that may affect the later stage of microRNA detection (PCR reaction).

Optimized scheme: For specific types of serum/plasma, according to different detection methods, optimization experiments with different volumes of starting samples should be performed first to determine the most suitable starting volume for this type of sample experiment.

It is extremely sensitive to hemolysis, and the released intracellular microRNA of blood cells will seriously dilute the original free microRNA.

Optimized scheme: The most abundant sources of blood cell contamination (red blood cells, platelets) can be selected as high-abundance microRNAs as reference materials for hemolysis quality control (commonly used are miR-451a, miR-23a-3p). By detecting the expression of these microRNAs, the degree of hemolysis can be evaluated.

Lack of stable and recognized internal reference.

Optimized scheme: Multiple microRNAs that are highly expressed in serum/plasma of healthy people can be selected as references. The commonly used ones are miR-30c-5p, miR-103-3p, miR-124-3p and miR-191-5p .

Q8: When studying peripheral blood microRNA, should we focus on the microRNA located inside the exosome or outside the exosome?

A: The selection of microRNA inside and outside the exosome as the research object should be determined according to specific factors such as the purpose of the research and the development conditions.

Focusing on microRNA outside of exosome, the advantage is that the content of microRNA is higher, it is easier to detect, the project operation cost is lower, and the disadvantage is that the test results are easily affected by many factors mentioned in Q8, the workflow stability is poor, and the operation The deviation between persons is relatively large.

Focusing on the microRNA in the exosome, the advantage is that the workflow is easy to standardize and quality control, the ability to resist interference from external factors is strong, the results are reproducible, and the biological significance is clear; the disadvantage is that the content of miRNA in the exosome is low, isolation and identification The experimental process is more complicated, the experimental facility requirements are high, and the project cost is relatively large. These disadvantages are great challenges for beginners.

(source:internet, reference only)

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