Quantification of microRNAs directly from body fluids
using a base-stacking isothermal amplification method
in a point-of-care device
Maggie R. Williams
Robert D. Stedtfeld
Tiffany M. Stedtfeld
James M. Tiedje
Syed A. Hashsham
Springer Science+Business Media New York 2017
Abstract MicroRNAs have been proposed to be a class of
biomarkers of disease as expression levels are significantly
altered in various tissues and body fluids when compared to
healthy controls. As such, the detection and quantification of
microRNAs is imperative. While many methods have been
established for quantification of microRNAs, they typically
rely on time consuming handling such as RNA extraction,
purification, or ligation. Here we describe a novel method
for quantification of microRNAs using direct amplification
in body fluids without upstream sample preparation. Tested
with a point-of-care device (termed Gene-Z), the presence of
microRNA promotes base-stacking hybridization, and subse-
quent amplification between two universal strands. The base-
stacking approach, which was achieved in <60 min, provided
a sensitivity of 1.4 fmol per reaction. Tested in various per-
centages of whole blood, plasma, and faeces, precision (coef-
ficient of variation = 2.6%) was maintained and comparable to
amplification in pristine samples. Overall, the developed
method represents a significant step towards rapid, one-step
detection of microRNAs.
Keywords MicroRNA quantification
Point-of-care (POC) nucleic acids-based methods and tech-
nologies have the potential to offer minimally invasive alter-
natives to biopsies and routine examinations allowing early
detection and therapeutics. MicroRNAs (short, non-coding
RNA molecules) are one such potential marker of disease
and cancer with varied expression levels in tissues (Esquela-
Kerscher and Slack 2006;Gauretal.2007), faeces (Link et al.
2012;Linketal.2010), saliva (Michael et al. 2010;Shaoetal.
2012), and blood (Schwarzenbach et al. 2014) between patient
samples and healthy controls; depending on the disease
(Ruepp et al. 2010). For example, increased expression of
miR-30b, miR-29b, miR-142-2p, miR-144, miR-203, and
miR-223 (> eight fold in some instances) has been observed
in oral squamous samples collected from cell carcinoma pa-
tients compared to healthy controls (Manikandan et al. 2016).
In serum, miR-141 expression levels up to 46 fold between
patients with prostate cancer and healthy controls (Mitchell
et al. 2008). Thousands of microRNAs have been identified
in humans (Griffiths-Jones et al. 2008; Griffiths-Jones et al.
2006;Griffiths-Jones2004) and efforts to associate
microRNAs to various types of cancer and disease is extensive
and ongoing (Ruepp et al. 2010).
Methods to quantify microRNA require superior limit of
detection, large dynamic range, and precision (Tricoli and
Jacobson 2007). Existing amplification-based methods for
measurement of microRNAs include stem-loop reverse tran-
scription polymerase chain reaction (RT-PCR) (Chen et al.
2005) and reverse transcription-free PCR (Lu et al. 2011).
Isothermal approaches such as rolling circle amplification
(Harcourt and Kool 2012; Liu et al. 2013;Zhouetal.2010),
loop-mediated isothermal amplification (LAMP) (Li et al.
2011), exponential amplification reaction (EXPAR) (Wang
et al. 2014; Zhang and Zhang 2012), and others have also
* Syed A. Hashsham
Department of Civil and Environmental Engineering, Michigan State
University, East Lansing, MI 48824, USA
Center for Microbial Ecology, Michigan State University, East
Lansing, MI 48824, USA
Department of Plant, Soil, and Microbial Sciences, Michigan State
University, East Lansing 48824, USA
Biomed Microdevices (2017) 19:45