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Luxembourg researchers discover significant lab kit contamination; team up with company to find solution



Blood, sweat & tears: This is the story of how a Luxembourg research group discovered the unlikely contamination of a widely used lab kit – compromising their data and setting a question mark over the validity of data in countless journal publications. We speak to group leader FNR ATTRACT Fellow Prof Dr Paul Wilmes about the implications of their discovery, reproducibility, scientific due diligence – and how his group teamed up with the kit manufacturer to find a solution.

The beginning

Rewind to 2011/12. Prof Dr Paul Wilmes and his research group Eco Systems Biology, based at the LCSB of the University of Luxembourg, develop a biomolecular extraction methodology. The group apply it to biomass from a wastewater treatment plant, local river water, and human stool samples. The stool showed apparent enrichment in Small RNA[1][2].

This got the team wondering if this small RNA may play a role in human-microbe interactions. It would make sense – it’s known that in particular microRNAs play essential regulatory roles in mammalian physiology. They have also been found to be dysregulated in e.g. cancer and neurodegenerative diseases, making them a great source for potential disease biomarkers.

“About 50% of the sequence data was not human, but from bacteria, archaea, fungi, food and even mosquito RNA”

Over the years, research has particularly turned to blood to try to find any markers that could reflect the diseased or healthy state. The issue here: there is little or no consistent or common line through many published studies in terms of results.

After Prof Wilmes and his team found Small RNA in human stool, they wondered if this could also be found in blood. In an initial study, teaming up with researchers in the US, the team looked at the blood – this is where the first unexpected results came in:

This finding suggested it may in fact be possible for Small RNA from the microbiota in the gut to travel into the bloodstream. Realising the potentially enormous insights this type of information could provide, Wilmes applied for a grant from the FNR’s PoC (now JUMP) programme to investigate further.

“It was clear that this could be an enormously valuable resource”

In addition to the PoC grant, Prof Wilmes had also applied for a patent for their biomolecular extraction methodology which has since been granted. Successful in getting the FNR grant, Wilmes and team got to work on getting more sequence data.

“We looked at the data using a new, more precise, bioinformatic pipeline we developed especially for the problem of accurately linking the short sequences to the organism of origin and this took 6 – 9 months. When we then looked at the list of identified organisms, it raised many questions.

Paul Wilmes – discover more about Paul Wilmes in our interview FNR ATTRACT Fellows – the people behind the science

“It’s for example not inconceivable that humans ingest quite a lot of fungi from contaminated food and therefore you might find RNA from these organisms in the blood.”

Testing to rule out contamination

At this point, Wilmes explains, they decided to test all sampling material and labware that comes into contact with the samples to rule out contamination:

Anna Heintz-Buschart did an enormous amount of “detective” work involving for example mock extractions, running the test with RNA-free water instead of blood, and that’s when it became clear that something was contaminated. But what could it be? The team painstakingly looked at everything and finally discovered that it was the RNA purification columns, specifically the silica in the columns. At this point, the team was nearly two years into the PoC project.

To verify the findings, the team used bleach on the columns, as it removes RNA, and then sequenced the eluate from the column again, confirming that the non-human RNA was already in the column long before they used it – putting an immediate end to their project of looking at RNA making its way from the gut to the blood.

The contamination was traced to the silica in the RNA purification columns

“We had a tough week when we found out about the contamination. We thought ‘how can this possibly be true?’”

Anna Heintz-Buschart, first-author on the paper explains: “Before we had the more accurate bioinformatics pipeline, we did not have a chance to validate the sequencing results on the blood extracts and we also could not search for the sources of the exogenous small RNAs in our environment. With the pipeline’s results, we could design independent, highly specific assays for these sequences – and test blood samples from unrelated individuals and the environment. We were very worried when we found the sequences in question literally in all samples we tested.

“In spite of the doubts we all shared, finding that the cornerstone of this project had essentially been missing all the time was a big shock. Fortunately, our crew had a very open, collaborative culture which meant that we discussed the implications of this finding and how we could deal with it in a constructive way,” says Wilmes.

“It was important for me that we come up with a solution instead of just leaving it at the stage where we knew there was a problem”

Wilmes contacted the manufacturer – Qiagen – to inform them of the findings and to find out if they would fund a follow-up study so the problem could be addressed.

With help of Prof Wilmes and his team, Qiagen have now developed a revised version of the kit – Wilmes’ lab has verified contaminants no longer play a role, according their analysis. Wilmes’ team has also come up with a whole suite of recommendations for future work to rule out contaminants playing a role, e.g. to use at least 100 microliters of blood plasma for RNA extraction.

The implications

The implications of the discovery go far beyond the project of Prof Wilmes’ group – this kit is one of the most widely-used RNA extraction kits – there are hundreds of publications, in some more than 99% of the data is from contaminating RNA.

The ongoing issue of reproducibility comes to mind: Could this contaminated kit help explain why there is little or no common line or constant in published studies looking at RNA biomarkers in blood?

Scientific due diligence

Asked about how he thinks things would have developed had they not checked for contamination, Wilmes explains that they might have published papers, but that they probably would have discovered the contamination at some point.

Contamination findings & recommendations published

A publication detailing the RNA extraction kit contamination was published online (open access) in the journal BMC Biology on Monday, 14 May 2018, with Anna Heintz Burschart as first author.

You can also read about the publication on the LCSB website.

Biomarker search continues

Wilmes and his group continue their scientific investigations into Small RNA potentially ending up in the bloodstream.

“From our work, we know that bacteria from the gut export small RNA. We have for example published a paper showing that this is the case for E. coli in part through packaging these into outer membrane vesicles. The key now is going to be to look at individuals where the barrier function of the gut is impaired, such as in inflammatory bowel disease and other diseases such as colorectal cancer. If there is this export of Small RNA into the blood due to a lack of barrier function that would be quite interesting from the point of view of blood-borne biomarkers for these diseases.”

[1] RNA = Ribonucleic acid, a nucleic acid present in all living cells. Its principal role is to act as a messenger carrying instructions from DNA for controlling the synthesis of proteins, although in some viruses RNA rather than DNA carries the genetic information. source

[2] usually non-coding RNA molecules, with the most common and well-studied example being RNA interference (RNAi), in which endogenously expressed microRNA (miRNA) induces the degradation of complementary messenger RNA – source


Prof Dr Paul Wilmes, Head of the Eco Systems Biology group at the LCSB at the University of Luxembourg