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Study Shows One Tenth of miRNAs From Stored Plasma Samples Meet Reproducibility Standards

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NEW YORK (GenomeWeb) – Circulating microRNAs are anticipated to become valuable medical biomarkers, but to date no one has assessed their stability after prolonged storage.

Now, researchers in Boston have demonstrated that a subset of plasma miRNAs in a panel associated with cardiovascular disease could be reliably measured after up to 14 years in liquid nitrogen.

The team — which includes researchers at Harvard's T.H. Chan School of Public Health, Harvard Medical School, Brigham and Women's Hospital, and Beth Israel Deaconess Medical Center — ultimately aims to determine if miRNA expression levels can be used to predict whether a currently healthy person will go on to experience cardiovascular disease in the future, Monica Bertoia, lead author on a PLoS One study published last week describing the research, told GenomeWeb in an email.

If the research can uncover whether higher-than-average or lower-than-average levels of a particular miRNA are associated with a greater risk of developing cardiovascular disease, this may elucidate the pathobiology as well as help identify individuals who are at a higher risk, Bertoia said.

To address this question, Bertoia and her colleagues used samples from a vast collection of plasma obtained as part of the Health Professionals Follow-up Study.

This ongoing all-male study compliments the all-female Nurses Health Study, and involves collecting health-related information as well as blood samples. It initially enlisted 51,529 men in health professions when it began in 1986, and has collected information regularly since. 

Blood samples were collected into EDTA tubes and frozen, then stored at the Brigham and Women's Hospital Harvard Cohorts Biorepository, in what amounts to a roomful of liquid nitrogen tanks.

In theory, this repository could be a treasure trove for a prospective cohort study, but there is little data on reproducibility of miRNA measurements from stored plasma, Bertoia explained, noting that there have been studies of other processing and storage conditions.

For example, one study found two miRNAs to be stable after boiling, sitting at room temperature for 24 hours, and multiple freeze-thaw cycles. Another study found three miRNA that were similarly stable, while still another found four miRNAs whose levels changed with varying conditions. 

But no study had yet looked at plasma that had been frozen and stored for such a long time.

Bertoia and her group examined 61 miRNAs that were plausibly related to cardiovascular disease in a representative subset of plasma samples from the HPFS cohort.

They performed RT-qPCR on the miRNAs and looked at inter-assay reproducibility, delayed processing stability, and short-term reproducibility of miRNA measurements within each individual over a period of about one year.

The later experiments used a set of samples from 40 healthy and 40 "less healthy" participants, and plasma that was stored for between 13 and 14 years.

Specifically, the group used an RNA isolation kit from Exiqon; the TaqMan MicroRNA Reverse Transcription Kit and two pre-amplification kits from Thermo Fisher's Applied Biosystems; and high-throughput RT-qPCR using the BioMark Real-Time PCR system from Fluidigm.  

Although housekeeping genes can be used to normalize other types of PCR data, there is no such thing as housekeeping miRNA, so "standardization of miRNA expression levels is more challenging," Bertoia noted.

The PLoS One study relied on a method called global mean normalization, as described in a 2011 Methods in Molecular Biology article, authored by some of the creators of the MIQE guidelines for PCR.

That method uses the difference between the expression level of a particular miRNA and the average expression levels of all miRNA measured within a given individual. "This can tell you if a particular miRNA is over- or under- expressed compared to average," Bertoia said.

The team also needed positive values to calculate coefficients of variation and intra-class correlation coefficients, so added back the average expression of all miRNAs from all individuals, or the standard global expression level, to the global mean normalized values in order to transform them.

The study ultimately found that only six of the 61 miRNAs — miR-17-5p, miR-191-5p, miR-26a-5p, miR-27b-3p, miR-320a, and miR-375 — met the group's inclusion criteria to be reliably used for cardiovascular disease risk prediction, but noted that querying larger sets of miRNA may still be needed for normalization purposes. And the field still lacks a method to normalize miRNA in studies with just a few candidate miRNAs.

"It is important that investigators aiming to examine prospective associations between miRNA expression and future disease risk pilot these biomarkers within their own system, unique study population, and sample collection and processing procedures to minimize the risk of futile studies," Bertoia said. 

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