Extracting Circulating microRNA, Part One: The Preanalytical Phase

microRNA (miRNA) is leading the way as circulating biomarkers in disease biomarker discovery. The hype around circulating miRNA is warranted. They meet many criteria for a good biomarker—namely, stability in circulation and varying levels in certain pathological conditions. Recently, studies have shown that miRNAs could be useful markers for cancer, myocardial infarction, atherosclerosis, and neuro-degenerative diseases.

This series of articles aims to provide you the best advice for extracting extracellular microRNA from blood. However, you must consider some important factors to consider before even starting the process. This article will cover the preanalytical phase. Our next article will delve a bit deeper and describe the advantages and disadvantages of different methods for microRNA extraction.

Stronger in Complexes: Circulating microRNAs

miRNAs are not found isolated in the blood. Instead, they circulate in complexes with Argonaute2 protein, lipoproteins, or within extracellular vesicles. These interactions explains their stability within the circulation. However, sample collection and preparation can effect the make up of these complexes—and thereby your choice of extraction method. Below are 6 important points to consider in sample collection and blood processing.

1. Choosing Between Serum or Plasma

Researchers isolate miRNA from both serum and plasma. However, serum is taken after blood clotting, which can result in some differences in the specific content of microRNA. These differences in miRNA content is due to release the of miRNA from platelets during their activation. Many studies, though, do not find significant differences between serum and plasma miRNA composition. Still, higher concentrations of miRNAs are consistently detected in sera.

2. Fasting Conditions

Should you draw blood from your patients before or after breakfast? Yes, food intake may influence the miRNA levels in the blood. Remember that miRNAs are carried in the circulation by high density lipoproteins (HDLs), among other particles. A patient can increase the amount of lipoproteins in the circulation just by eating. This may then have an influence on the efficiency of miRNA recovery. Just to be sure, do any blood draws (phlebotomy) before the patient eats (preprandial).

3. Controlling for Hemolysis

Rupture erythrocytes (hemolysis) can leak miRNAs into the circulation and might alter miRNA levels or the composition of the miRNome. For example, ruptured erythrocytes can release miRNA, which is especially problematic, because it is often used as an endogenous control. Apart from changing the miRNA levels, hemolysis also causes the release of hemoglobin and lactoferrin. Both compunds negatively influence PCR reactions.

The first, and best way, to minimize hemolysis is to work with a well-experienced phlebotomist. Next, carefully select the correct conditions for sample storage and transport. For example, do not freeze the samples upon blood draw—rather keep them in the fridge before processing. Also, avoid vigorous mixing of the tubes (because this can also cause hemolysis).

Even with all of the precautions above, it is hard to avoid some lysis of erythrocytes. So, you should always perform a hemolysis test. Exclude any samples that do not pass this test. A very simple hemolysis test is the Harboe direct spectrophotometric method. This involves a 3-point absorbance measurement at wavelengths of 415, 380, and 450 nm. A more complicated, but more sensitive test, is the measurement of miRNAs as haemolysis indicators, such as the miRNA-451a to miRNA-23a-3p ratio.

4. Checking Blood Cell Counts

The peripheral blood cell count also complicates miRNA profiling from blood. An increase in the number of platelets that get activated during venipuncture can mess with the miRnome. Also, even if you’ve taken precautions to avoid hemolysis, erythrocytes can release some miRNAs during longer storage periods. Unfortunately, there is little you can do about it if you are not in a position to exclude samples with higher blood cell counts. Make sure, however, to have these data so you can explain any odd results in case they appear.

5. Using the Appropriate Tubes

There are a number of different tubes you can use to collect blood. These differ in the content of conservation medium and anticoagulant. So which ones do you use?

This decision depends on two things: (1) the timeline of your blood processing and (2) whether you use serum or plasma.

If you cannot process the samples immediately, you must use tubes that have an RNA conservation medium. This will protect the miRNA from degradation. Such tubes enable the storage of whole blood samples for several days in the refrigerator. If you can process the samples within a few hours (careful not to wait more than 6 hours!), then you can use regular tubes.

Next, you still need to decide if you want tubes with or without anticoagulant. This, of course, depends on whether you collect serum or plasma.

As you might expect, not all anticoagulants are appropriate. For example, heparin inhibits the activity of reverse transcriptase and polymerase. Citrate can dilute plasma, which causes hemolysis because of hypotonicity. It also interferes with any downstream PCR applications. The best choice for an anticoagulant is EDTA because it is less likely to interfere with downstream PCR applications or cause hemolysis.

6. Storage Conditions

Finally, you are ready to store your samples! (Ok, first, you must remove the blood cells from plasma or blood clots from serum. And do another spin at 2500 g for 15 mins to get rid of the remaining platelets in your plasma. But after that, you’re ready!)

Yes! That means you do not have to stay in the lab until the wee hours of the morning to do the RNA extraction. You can aliquot and store your samples at -80°C. Reassuringly, according to most studies, freezing and thawing of miRNA does not affect the stability and recovery of miRNAs.

Well that is a lot of info! Stay tuned for the next article in this series that covers the methods of miRNA extraction!

Image Credit: National Institute of Health

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