Determining the quality of biobank serum for microRNA analysis miRCURY LNA™ Universal RT microRNA PCR
Dr. Wendy Alderton
is a spin out from University College London started 2 years ago and has an exclusive commercial license to a large prospective serum biobank. The company provides access to this biobank and through collaborations with commercial diagnostic partners, academic groups and not-for-profit organizations, advances biomarkers for the early detection and screening for cancer. In December 2012 Abcodia was awarded ‘NatWest Startups Business of the Year’. Abcodia has recently completed a successful pilot study on microRNA detection with Exiqon services which confirms the the quality of the UKCTOCS serum for microRNA discovery and validation projects.
What is the serum biobank?
Between 2001 and 2005, over 202,000 women volunteers were enrolled into the United Kingdom Collaborative Trial of Ovarian Cancer Screening (UKCTOCS) which was led by Abcodia's academic founder Professor Ian Jacobs at the Institute for Women's Health at UCL. Volunteers donated a serum sample on trial entry and up to 50,000 women have continued to donate serum annually for up to 10 years. The serum was collected to a rigorous protocol and is stored in liquid nitrogen at a commercial biobanking facility at Fisher BioServices. The women were all cancer free at the start of the trial however, they have gone on to develop a whole range of diseases including >27,000 cancers as well as other conditions. The availability of such large numbers of longitudinal pre-diagnosis samples, together with demographic and disease data that allows the identification of all major age-related diseases in this cohort, makes this an ideal resource for investigations relating to screening diagnostics. The cohort allows us to match healthy volunteers, with benign and in situ
tumors as well as other diseases that are known to interact with a potential biomarker. Abcodia's serum biobank
When and how did you come to be interested in looking at microRNAs as biomarkers in serum?
Abcodia has been investigating the full range of molecular entities that could be quantifiable in the UKCTOCS serum and validated as early detection biomarkers for a range of cancers. We have been aware from the time the company was first founded of the potential of microRNA biomarkers due to the extensive literature in this area on both tissue and blood biomarkers studies. The stability of microRNA in serum makes it an attractive area to study but we wanted to independently confirm this and also identify the most appropriate technical platform.
What is the aim of the current project?
The study we undertook was a small pilot study design to build confidence in the quality and applicability of the UKCTOCS serum samples to future larger microRNA biomarker discovery projects. The serum samples in the biobank have an average time to spin from venepuncture of about 20 hours during which time they were stored at ambient temperature. Therefore, we wanted to assess the stability of the microRNA signals and whether there had been significant hemolysis during this time which may potentially contaminate samples.
Which experiments had you performed leading up to this project?
Prior to this experiment we had lots of experience of other markers, such as proteins, auto-antibodies and microRNA but not using the qPCR methods employed by Exiqon.
How did you perform the pilot experiment and analyze the results?
This pilot study used serum samples from 20 cases of one cancer type, taken at two time points one year apart and matched controls, giving 80 samples in total. We profiled these samples against microRNAs which have been identified in the literature as being upregulated in plasma or serum as well as Exiqon’s standard QC controls giving 29 microRNAs in the test set. The normalization of the data was particularly challenging since this was such a small study there were limited options for the normalizers. This is an area we are still investigating and reading around more from the literature on alternative normalization procedures.
How do you feel about your results so far?
We were very pleased with the results of our pilot study as the data provides confidence in the quality of the UKCTOCS serum for future, larger microRNA discovery projects. The data from the no template and RNA spike in controls indicated a good technical performance of the sample processing. Only two samples from the total of 80 samples were judged to be potential outliers in a principal component analysis and were excluded from further analysis. No signs of hemolysis, which may contaminate samples with microRNA from red blood cells, were found in the samples using the Exiqon’s standard method for hemolysis detection. On average, 80% of the microRNA candidates profiled in this experiment were detected in the samples and 50% of the microRNA candidates were detected in all samples.
What were some important aspects for you when choosing a microRNA service partner?
We would look for an experienced microRNA service partner who can provide a high-quality service in a reasonable timescale at competitive prices. It is also important to us as relative newcomers to this field that interpretation and explanation of the data is included as part of the service so we can fully understand the results and the implications for future studies.
Would you recommend Exiqon and/or Exiqon’s microRNA qPCR system to colleagues, and if so, why?
We would certainly recommend Exiqon’s microRNA qPCR system to colleagues and potential collaborators and in fact have already done so. The services were completed professionally, efficiently and to the agreed timescales. The analysis report was detailed and clearly explained. Exiqon staff were also extremely helpful in helping to design the study, analyze and interpret the results for us using all their past experience in this area. The scientists were also very knowledgeable and helpful in answering some of the questions we had on the basic biology and normalization methods since we are relatively new to this field.
What would be your advice to colleagues about getting started with microRNA profiling and biomarker discovery?
There are several different platforms available for the detection of microRNA and we feel that it is important to understand the likelihood of each platforms as suitable for the biospecimen being used. The approach we took and would recommend to others is a small pilot across multiple platforms before embarking on a major discovery experiment.
Identifying the microRNAs that you would expect to discover during this pilot, for example, we would recommend through a thorough reading of the literature to understand what has previously been identified provides a basis for comparison with your results.
As well as the technology platform one should also consider the capabilities of the service provider in performing larger scale studies in a timely and cost effective way. We found Exiqon to be an experienced and capable partner.
What are the next steps in the current project and how do you plan to perform them?
Having gained confidence in both Exiqon’s LNA™ microRNA qPCR system, the excellent service they provided and the quality of the data generated so far we would like to embark on larger studies using Exiqon’s Human PCR Panels (for detection of 742 microRNAs) in a number of cancers to discover novel, early detection biomarker panels. The unique nature of the longitudinal, pre-diagnosis samples we have access to make them ideal for identifying screening biomarkers which would diagnose cancer earlier thereby greatly increasing the chances for successful clinical management at stages before the establishment of metastatic disease.