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Extracting Circulating microRNA, Part Two: Isolation Methods

In the second part of this two part series on analyzing extracellular microRNA (miRNA) from blood or serum, we continue with the methods for miRNA extraction.

Generally, there are two different approaches for microRNA isolation. The first of these is organic, liquid-liquid. The total RNA extraction method employs phenol/chloroform isolation with guanidine isotiocyanate as a denaturant. The second is column-based solid phase extraction methods that can be modified to catch only small microRNAs. That being said, the column-based methods often employ organic extraction as a separation step.

Organic Extraction

One usually does organic extraction using Trizol/Qiazol commercial reagents. A specific Trizol reagent is designed for liquid samples. The basic difference is in the concentration of the components. The procedure accommodates the larger volumes that one typically uses for liquid samples (usually around 200 µl of serum/plasma). However, if you happen to have a full stock of regular Trizol, it works quite well to just increase the volume of the reagent. In particular, the ratio should be around 1:5, sample to reagent. Some authors recommend even higher ratios, such as 1:10, to ensure proper denaturation of proteins.

Advantages of Organic Extraction Methods for miRNA

Organic extraction is not expensive. Also, you get a higher yield of RNA and lower inter-assay variability. Furthermore, you can store samples in Trizol if you don’t have enough time to process them. Some authors even recommend this to help completely denature the proteins in the plasma. Trizol is also a good option for samples with category A pathogens. These must be inactivated using guanidinium and phenol solutions.

What Carrier Should You Use for Extracting miRNAs?

It’s a good idea to use a carrier for precipitating microRNAs. Researchers have conflicting opinions about the use of tRNAs and MS2 phage RNA for this purpose. Some authors consider these to be responsible for nonspecific hybridization or amplification in downstream applications. Therefore, a safer option is to use glycogen as co-precipitant. Glycogen is free of biological contaminants and does not interfere with PCR.

Precipitation Time

A special recommendation for miRNA isolation with Trizol is in the precipitation step. You should increase the precipitation step to overnight at -20°C. Some studies recommend that following this step that a higher speed and longer time should be used for pelleting the RNA (16k – 20k g, for 1h). So, add the carrier and the isopropanol. Then, store the samples in the freezer to increase the chances for small RNAs  precipitating.

Disadvantages of Organic miRNA Extraction

The biggest problem with Trizol is the purity of the sample when you measure its A260/230 ratio. Phenol/chloroform extraction leaves the samples with organic solvent (phenol, guanidine isotyocyanate) contaminants. These can inhibit PCR. Given the small amount of miRNAs in circulation, this contamination may have a significant influence.

Column-Based Methods

Because of the contamination issues with organic extraction, and the fact that it is a time consuming technique, column-based methods are a great alternative.

A large number of commercial kits use a phenol/guanidine step for separating RNA from the rest of the material. A second step purifies the RNA through glass-fibre or silica-membrane filters. This results in greater purity of the sample than just using organic extraction alone. There are also kits that completely avoid this separation step. They use proprietary lysis buffers and  resins to extract the RNA instead.

Advantages of Column-Based miRNA Extraction Methods

Column methods are very fast, which makes them a favorite. MicroRNAs are recovered in solid amounts and ran through columns to clean them up. So, even if the extraction includes an organic separation step, the final sample is free of organic contaminants. Studies show that you can isolate a larger variety of miRNAs using solid phase extraction. So, if your main goal is to profile the microRNAs in the sample, columns are way better way to go. Reproducibility, is also better. So that’s another plus for solid phase extraction!

Small miRNA enrichment

Some commercial kits have an option to use a modified step for isolating small microRNA. These include a stepwise increase in the amount of alcohol used for precipitation. However, if you are working on profiling microRNA, it is better to isolate total RNA. That way you can critically evaluate RNA quality and quantity to see if the sample is suitable for downstream microarray analysis.

Disadvantages of Column-Based miRNA Extraction Methods

The most obvious disadvantage of column methods is their higher price in comparison to liquid extraction. Most researchers also complain they get lower yields and the variability between samples is higher.

Furthermore, columns can clog when using larger volumes of the starting material (over 200 µl). Proteins or particle debris are the main culprits for this.  Another issue is the capacity of the membranes, which can sometimes be suboptimal. This is because they tend to saturate with larger RNA species. So be careful with the amounts of starting material.

You will need a need a specific kit for isolation if you are collecting the blood in tubes with conservation medium. This is because these systems are designed for each other.

Quick Tips for miRNA Extraction Methods

Because a bunch of recommendations and opinions are found in these two articles on circulating micro RNA extraction, here’s the short take-home points:

  • Sample preparation is super important: use pre-prandial blood, check for haemolysis and check for the platelets/erythrocytes count.
  • Use EDTA anticoagulants if you process the blood immediately. Use conservation tubes if samples need to be stored for a few days.
  • Freeze aliquots of serum/plasma at -80°C; feel free to freeze/thaw several times.
  • Use organic extraction if you are measuring a defined set of miRNAs due to higher yields, but opt for kits if doing profiling.
  • Report all the details of sample collection and preparation in the papers.
  • And, as usual, whichever protocol you use, stick to it, and process the samples in the same way!

We encourage you to write your experiences in the comments section since this is an underexplored field. Any piece of advice or experience will be very useful for all of us!

Good luck!

Image Credit: National Institute of Health

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