Quantification of microRNAs in body fluids miRCURY LNA™ Universal RT microRNA PCR
Dr. Florian Kuchenbauer
, Group leader and Sarah Grasedieck
, PhD student, study acute myeloid leukemia at the University of Ulm in Germany. They have been using Exiqon's miRCURY LNA™ Universal RT microRNA PCR system.
1. What is the main focus of your research?
We try to decipher genetic and epigenetic mechanisms of lineage fate decisions in normal and aberrant hematopoiesis with special interest in acute myeloid leukemia (AML). In addition, we investigate the role of non-coding RNAs in AML and the potential of extracellular microRNAs as novel biomarkers for different diseases.
2. What is the aim of your current project?
Our current project involves the analysis of extracellular microRNAs in body fluids. The overall goal is to develop diagnostic microRNA tests for malignant and non-malignant diseases. In addition we hope that our studies may also lead us to understand the functional aspects of blood- and cerebrospinal fluid (CSF)-born microRNAs.
3. How did you approach your studies of extracellular microRNAs?
For the work with and profiling of extracellular microRNAs, a variety of different strategies are reported in literature. To analyze miRNAs in a consistent and comparable way, we started our research by getting to the bottom of basic principles like the most efficient way of microRNA extraction from body fluids. We also investigated the influence of external factors on microRNA integrity, e.g. the storage conditions of patient samples. In this regard, we also tested different quantification methods and decided to stick with qPCR in our experiments.
4. What were some specific challenges in your experiments and how did you overcome them?
One of our main challenges was to reliably detect microRNAs in CSF. The amount of sample available is limiting and the levels of microRNA present are very low. Firstly, it was very important for us to optimize our RNA extraction procedure, and secondly, it was important to have a very sensitive and reliable microRNA qPCR system.
5. Why did you choose to use Exiqon’s miRCURY LNA™ microRNA PCR system?
From the small sample amounts that were available to us, we wanted to obtain as much information as possible. During our initial investigations we have worked with different microRNA qPCR methods. Generally our experiences were very positive, although other qPCR approaches, were very elaborate and sample-consuming. For our initial studies, we found that the Exiqon Focus panels provided a quick and easy solution .
6. What do you find to be the main advantages of the miRCURY LNA™ microRNA PCR system?
When working with very low miRNA concentrations as found in CSF, the main advantages of the Exiqon microRNA PCR panels are found in their high sensitivity and low amounts of precious starting material needed.
7. How do you feel about your results so far?
After having spent some time optimizing our experimental procedures, we are very confident about our data so far.
8. What would your advice be to colleagues wanting to get started with the study of extracellular microRNA?
It is very important to make sure you are getting the most out of your samples. Compare different extraction protocols and perhaps make use of synthetic microRNA spike-ins to control the constant efficiency of your RNA extraction procedure and RT reactions. For initial microRNA profiling, it is important to compare different qPCR protocols and eventually stick to one approach. Sample selection can also be critical. In our studies we found that even after four years of sample storage at -20°C, the overall amount of serum microRNA levels is barely changed and shows only minor differences in the levels of individual microRNAs (Grasedieck et al., Leukemia, 2012). However, for the accurate quantification of single microRNAs, storage conditions should be kept constant within a cohort of samples and samples for retrospective analyses should be chosen according to this criterion.
9. In your opinion what is the most important factor for a successful microRNA qPCR experiment?
For us, it was important to try various techniques for RNA extraction and microRNA profiling approaches. This allows you to develop a feeling for your samples and experiments. Be consistent with your controls and use more than one way to normalize your samples to test the consistency of your results.
10. What are the next steps in the current project?
With Exiqon’s Human PCR Panels (for detection of 742 microRNAs), we plan to identify diagnostic microRNAs and subsequently establish novel diagnostic tools.
11. What are the future perspectives for this research?
The generation of a diagnostic platform that covers several areas of disease and easily integrates into daily clinical routine – from bench to bedside.
12. When will we hear more about your exciting studies?
Original publications will follow soon.