Abstract
Introduction
MicroRNAs (miRNAs) are involved in the control of hematopoiesis, their deregulation also appears to play role in the pathogenesis of several hematopoietic diseases. Multiple miRNAs have been reported to be abnormally expressed in hematologic cancers; moreover, specific miRNA expression profiles have been proposed as diagnostic and prognostic markers in various hematologic malignancies, including Myelodysplastic Syndromes (MDS). MDS are a heterogeneous family of clonal disorders of hematopoietic stem cells. They are characterized by ineffective hematopoiesis and frequent leukemic progression.
It has been shown that the mature erythrocytes are rich in diverse miRNAs species, even though they lack ribosomal and other large-size RNAs. miRNAs are expressed during erythrocyte differentiation and have an important role in erythropoiesis and mRNA degradation.
Methods
Four miRNAs, i.e. miR-16, miR-181, miR-34a, and miR-125b were selected as a model set of potential MDS biomarkers and evaluated using a surface plasmon resonance imaging (SPRi) biosensor and real-time PCR. The sensor calibration curves were measured with miRNAs spiked in a complex erythrocyte lysate. The total RNA was extracted from erythrocyte lysate using the acid guanidinium-thiocyanate-phenol-chloroform method. The levels of selected miRNAs were measured in all samples by quantitative reverse transcription - real-time polymerase chain reaction.
Results
A high-capacity array SPRi system for rapid simultaneous detection of multiple miRNAs in erythrocyte lysate was developed and demonstrated at the Institute of Photonics and Electronics. The ultra-low fouling functionalizable poly(carboxybetaine acrylamide) (pCBAA) brushes-coated gold surfaces of the SPR sensor were shown to reduce the non-specific interaction between surface of the sensor and sample. A two-step miRNA detection assay for multiplexed miRNA detection in erythrocyte lysate was demonstrated in which hybridization of probe-functionalized pCBAA with target miRNA bound to biotinylated oligonucleotide probes was followed with capture of streptavidin-functionalized gold nanoparticles to biotinylated probes. In preliminary experiments, the array was shown to be capable of detecting multiple miRNAs spiked in erythrocyte lysate without the need for complex lysate sample pretreatment at concentrations as low as 0.5 pM in less than 45 minutes.
Conclusions
Using the newly developed high-capacity array SPRi system we have found significantly increased levels of miR-16 in erythrocyte lysate samples (at the concentration range of ~10-100 pM) compared to other miRNAs tested within this study. The results were confirmed by a reference real-time polymerase chain reaction method. In addition, our results indicate an over-expression of miR-16 in erythrocyte lysate samples of MDS patients with both Refractory Cytopenia with Multilineage Dysplasia (RCMD)and Refractory Anemia with Excess Blasts (RAEB) diagnosis. This clearly demonstrates the potential of SPRi array technology for clinical applications.
Disclosures
No relevant conflicts of interest to declare.
The design, synthesis and characterization of a novel acceptor for real time polymerase chain reaction using both computational and experimental approaches
