Scientists around the world are searching for reliable biomarkers to diagnose diseases and track disease progression and patients’ responses to treatment. There has been a lot of excitement about the potential for circulating microRNAs to be biomarkers for a range of different diseases including cancer, cardiovascular disease and Alzheimer’s disease1. Viral microRNA has also been recovered from human bodily fluids. Therefore, viral microRNAs may be useful in diagnosing infectious viral diseases.
MicroRNAs are short, 18-22 nucleotide, non-coding RNAs that bind to target messenger RNAs (mRNAs) and regulate their expression. Circulating microRNAs are released into bodily fluids such as the blood and have been proposed as biomarkers because they are more stable than mRNAs. However, many studies have shown that different microRNAs have different stabilities at room temperature or at 4°C. Some are stable in serum or plasma samples stored at room temperature for at least 24 hours. Other microRNAs rapidly degrade when stored at 4°C.
Biobanks and laboratories often store serum samples at 4°C for short periods of time before analyzing the samples or moving them to -80°C or cryogenic storage. Two recent studies show that this practice may lead to significant degradation of some microRNAs, while other microRNAs will be unaffected.
MicroRNA-122 and MicroRNA-145 Rapidly Degrade at 4°C
A pilot study by scientists at Kyorin University in Tokyo, Japan looked at the stability of microRNA-122, microRNA-145 and microRNA-92a in serum stored at 4°C for 24 hours2. All three of these microRNAs have been proposed as biomarkers for various diseases. Circulating microRNA-122 is a possible biomarker for liver injury. MicroRNA-145 is a potential biomarker for cardiovascular diseases. Circulating miR-92a is a candidate biomarker for lung biomarker.
The researchers measured levels of all three microRNAs in the serum and in extracellular vesicles. Cells use extracellular vesicles to deliver microRNAs to other cells. The authors found that microRNA-122 and microRNA-145 rapidly degraded when stored at 4°C. Serum stored at 4°C for 24 hours had lost 64% of its microRNA-122 and 71% of its microRNA-145.
In contrast, microRNA-92a did not degrade in serum stored at 4°C for 24 hours.
MicroRNA-1 and MicroRNA-432-5 are Stable at Room Temperature
A separate study by researchers at the University of Sao Paulo in Brazil looked at whether microRNA levels can be affected by the amount of time that blood sits at room temperature before being processed.
If blood samples have a significant amount of hemolysis, microRNAs can escape from lysed cells and contaminate the serum or plasma component. This contamination makes it impossible to accurately measure the levels of circulating microRNAs.
The Brazilian study incubated ten blood samples for 0, 3 and 24 hours at room temperature before processing the samples to isolate plasma. Even though some of the samples showed significant hemolysis as measured by UV spectroscopy after 24 hours, they found minimal changes in microRNA-1 and microRNA-432-5 levels3. They also found no correlation between the amount of hemolysis and microRNA-1/microRNA-432-5 levels.
These two studies highlight the variability in circulating microRNA stability. Some microRNAs are stable in whole blood, plasma or serum samples at room temperature for at least 24 hours. Others degrade rapidly even when stored at 4°C. Therefore, to maintain the quality and integrity of all potential biomarkers within a sample, that sample should be frozen as soon as possible and temperature excursions during shipping, handling and storage should be avoided.
- Anfossi et al. Clinical utility of circulating non-coding RNAs – an update. Nat Rev Clin Oncol.
- Aiso et al. Degradation of serum microRNAs during transient storage of serum samples 4°C. Clin Biochem, 2018.
- Borges et al. Impact of Delayed Whole Blood Processing Time on Plasma Levels of miR-1 and miR-423-5p up to 24 hours. Microrna, 2018.