Quantifying circulating microRNAs in biomarker research can be a tricky business, according to a group of researchers who published a report last week highlighting a host of sample-collection and -preparation factors that can influence the accuracy of miRNA quantitation in studies and diagnostic tests.
In a study published Dec. 7 in the Journal of Molecular Diagnostics, the team described a variety of confounding factors they say significantly affects the accuracy of miRNA quantitation, such as the type of collection tube used in sampling, the influence of added anticoagulants and stabilizers, sample size, and the role of endogenous serum factors that co-purify with RNA and inhibit polymerases used to selectively amplify miRNAs in quantitative PCR assays.
The researchers reported that making several changes in their sample-collection and –preparation methods allowed them to improve the sensitivity of miRNA detection in their tests up to 30-fold in some cases. Such a dramatic increase highlights how much variability in sampling and preparation protocols can influence miRNA detection, Dominik Duelli, a molecular pharmacologist at the Chicago Medical School at Rosalind Franklin University and lead investigator of the study, told Gene Silencing News.
"Typically what you hear people say these days is, 'microRNA quantitation in the plasma is trivial,' and I agree. It is extremely trivial to get data," he said. "But for that data to actually reflect what the microRNA concentration is in the blood is absolutely not guaranteed unless you take [some] steps … and [address] the variables we identified."
Duelli said his group became interested in optimizing and standardizing quantitative RT-PCR miRNA detection after beginning research into circulating miRNAs as potential biomarkers in breast cancer.
There are two key issues in blood-based miRNA biomarker research, he said. "First ... not all microRNAs may be released [from cells into the blood], and second ... for some reason you don't see all” that are.
Several companies, including Life Technologies and Exiqon, now offer products for detecting miRNA in blood samples for biomarker development and disease research. With qPCR currently the most popular approach, according to the study authors, the field needs assurance that measured concentrations actually represent the amount of miRNA in a sample, and "such assurance is often lacking," Duelli and his colleagues wrote in their paper.
Using qPCR-based techniques and considering the factors highlighted in the paper could enable quantitation of low-abundance miRNAs when usual techniques fail, the researchers wrote, and could "increase the repertoire of miRNAs that can be analyzed as potential biomarkers of disease."
In investigating sample collection and endogenous co-purifying factors that can confound miRNA quantitation, the group quantified two miRNAs, miR-16 and miR-223, in blood samples collected from a number of sources, including healthy adults, and samples from AIDS and cancer patients.
The researchers tested a variety of sample-collection tubes, some without anticoagulants and others containing anticoagulants like EDTA, heparin, sodium citrate, or NaF/KOx. They also evaluated whether separately adding anticoagulants and blood stabilizers could offer additional benefit.
They reported the best results using tubes containing NaF/KOx. Though both miRNAs have very different abundance in blood, recovery and accurate detection for both of them depended on the blood-collection method, suggesting that collection procedures should influence additional miRNAs regardless of their abundance.
Tubes containing heparin, which the group used when beginning its research, are a bad choice, Duelli said, because heparin binds RNA and can interfere with polymerases. But if researchers are limited to heparin tubes, his group found that adding the heparin-digesting enzyme heparinase to the samples can increase the ability to detect miRNA.
Additionally, adding NaF and KOx later in the process can also benefit detection. Added together, the anticoagulants increased miR-16 detection two-fold in plasma collected using EDTA tubes and three-fold in serum, the researchers wrote.
With the influence of sample collection methods in hand, Duelli's group then went on to investigate issues in the composition of blood samples themselves that can interfere with qPCR, testing a variety of approaches to improve the purity of isolated RNA and increase the sensitivity of miRNA detection.
The first approach was to "just purify the plasma really well," Duelli said. However, repeated extraction and purification steps can become expensive and time-consuming. As such, the group profiled additional polymerases that might avert interference from blood-borne inhibitors without the need for repeated purification.
The researchers decided to try a Taq polymerase mutant, Hemo KlenTaq, developed by a team at Washington University School of Medicine in Saint Louis, to resist the binding of inhibitory molecules.
Unfortunately, in altering the polymerase to resist inhibitory molecules, the Washington University group had to remove a fragment responsible for effective proofreading of target sequences, which can also interfere with quantitation.
To overcome this issue, Duelli's team took a combinatory approach, adding an intact Taq polymerase to sway the reduced proofreading activity of Hemo KlenTaq. "We thought, 'Why not combine both worlds, take the polymerase that they developed to be really good for blood, and add a regular polymerase,'" he said.
"You get the huge amplification from the Hemo KlenTaq, but at the same time adding the normal polymerase, which is partially inhibited, can really help in reducing the background amplification of non-specific RNA," Duelli said.
The researchers then tested whether all the steps they used to overcome endogenous and sampling-related inhibitors would allow higher miRNA quantitation, analyzing circulating miRNAs in six healthy subjects.
"We looked at the overall abundance of these two microRNAs that we [most often] look at," Duelli said, "and what we found is, in some cases, it's very linear, very clear: in five out of six people, you get up to 30-fold more amplification products, so you increase the sensitivity and quantity quite a bit."
However, he said there was one subject where the increase wasn't equivalent. This suggests that differences in plasma composition from individual to individual can yield different miRNA measurements.
"MicroRNAs as diagnostic and prognostic markers have just taken off like crazy and I think for good reason. But I think we have to be very careful that we can validate these things," Duelli said.
"As far as I can tell, [when] you go from one lab to another things are not as standardized as proteins and mRNA, and that's because the field is still relatively young with a lot of excitement."
Duelli suspects many others have hit on some of the same issues and solutions his group presented in its paper. "I'm guessing other people know similar stuff, but I'm hoping the paper will help people who are just starting; or, for people who have done it for a while, maybe they will think to try some of this."
Because not all the samples from the six healthy subjects that the group tested showed the same level of improvement with the optimized sampling and preparation protocols, Duelli said, it is likely there are other blood components that the group was not able to account for, perhaps even individual-specific factors like age and habits.
In that case, "what could help is really just to standardize the blood-collection practices," he said. "For epidemiological studies, these things may even themselves out," but they may matter for the individual studies.
In their own research, Duelli said his team will plan to use NaF/KOx tubes, and the combination of Hemo KlenTaq and regular Taq polymerase. In the paper they rate all of the possible steps in terms of time, sensitivity, and cost, to allow other groups to pick and choose a path based on their own needs.
Duelli did not suggest that his study invalidates qPCR technologies currently being used for miRNA biomarker research. Many groups may have come to include the steps his team recommends on their own, he said. However, he noted that standardization across the field is still necessary.
Duelli said his team is also investigating another approach to bypass the issue of confounding co-purifiers in blood altogether by narrowing their miRNA analysis to molecules within specific nanovesicles associated with breast cancer cells. He said they plan to publish this research soon.
"We isolated, very specifically, microvesicles containing the miRNAs of interest and fished out specifically nanovesicles from breast cancer cells and quantified those," he said.
"If all the cells in the body release these vesicles and miRNAs, your blood is a complete soup of all kinds of stuff, and while people very clearly have found specific miRNAs that are up-regulated in cancer, as far as I know, almost none of them are specific to one type of cancer."
"So by going clearly after nanovesicles that we know by surface markers specifically came from, in our case, breast cancer, we can become more specific," he said.
This also overcomes issues in blood sampling, because the vesicles can be isolated using antibodies, said Duelli. "You avoid the entire problem completely because if you have an antibody against your specific vesicle, you remove all this other stuff."
This approach is very selective, however, rather than widely profiling circulating miRNAs.
Many researchers “are still going to need to look at the whole blood plasma," Duelli said. For that, the group hopes its study of confounding factors will prove useful.
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