The development of biomarkers will be essential for the future of diagnostic, prognostic and predictive medicine. Diagnostic biomarkers may be used to detect cancer, heart failure, stroke or a number of other diseases. Prognostic biomarkers predict patient outcome, while predictive biomarkers are essential for determining whether a cancer patient will respond to targeted therapy. Unfortunately, the translation of biomarkers from the bench to the clinic remains difficult (1). Collecting high-quality samples in sufficient numbers is one roadblock to biomarker studies. Biofluid-based microRNA profiling presents a readily available source of stable biomarkers. microRNAs, small 21 to 24 nucleotide regulatory RNA molecules, have been detected in a wide range of body fluids, including serum, plasma, cerebrospinal fluid (CSF), urine and saliva (2). They are highly expressed in serum and plasma and can be obtained in a minimally invasive manner. In this post, we explore methods for isolating microRNA from biofluids as well as techniques for microRNA profiling.
An attractive property of circulating microRNAs is that they are relatively stable (3). Circulating microRNAs are protected from RNase degradation by several mechanisms. One mechanism of protection is microRNA packaging in exosomes and microvesicles. Exosomes are abundant in plasma and are also present in serum, CSF, urine and cell culture media. Exosomes can be purified by ultracentrifugation or column-based methods, followed by isolation of total RNA or microRNA. Column-based exosomal isolation is generally fast, robust and easily automated. Although exosomes are a rich source of functionally relevant circulating microRNA molecules, only approximately 10% of extracellular microRNA is packaged in exosomes (4). The remaining 90% is protected from degradation via association with proteins such as Ago2 and HDL. Therefore, circulating microRNA can also be isolated from most body fluids using column-based methods or standard kits.
Once microRNA has been isolated, microRNA profiling can be accomplished using qRT-PCR, microarrays or RNA sequencing. Each method presents advantages and disadvantages. qRT-PCR is well established, sensitive, quantitative and can be performed easily in most labs. However, it will not identify novel microRNA molecules. Microarrays are also well established but have lower sensitivity than RNA-seq and cannot be used for absolute quantitation. RNA sequencing is accurate and sensitive but requires specialized expertise for data analysis. For biomarker discovery, researchers may wish to use a combination of techniques in sequence, such as large-scale microRNA profiling to identify expressed microRNAs followed by quantitative determination of differentially expressed microRNAs. Additional samples can then be tested using a panel of differentially expressed microRNAs for validation of predictive biomarkers. Regardless of the workflow, biofluid microRNA profiling has the potential to improve molecular diagnosis and predict therapeutic efficacy in a wide range of human diseases.
Interested in learning more about microRNA profiling in biofluids? Dr. Jonathan M. Shaffer presents an integrated system for circulating biomarker discovery from Sample to Insight. Advanced techniques for exosomal microRNA isolation and microRNA detection are also discussed. Check it out now!
- 1. Drucker, E. and Krapfenbauer, K. (2013) Pitfalls and limitations in translation from biomarker discovery to clinical utility in predictive and personalised medicine. EPMA J. 4, 7.
- 2. Etheridge, A. et al. (2011) Extracellular microRNA: a new source of biomarkers. Mutat. Res. 717, 85.
- 3. Blondal, T. et al. (2013) Assessing sample and miRNA profile quality in serum and plasma or other biofluids. Methods 59, S1.
- 4. Zhu, H. and Fan, G.C. (2011) Extracellular/circulating microRNAs and their potential role in cardiovascular disease. Am. J. Cardiovasc. Dis. 1, 138.