miRNA

Label-free Multiplex Detection of MicroRNAs

Introduction

MicroRNAs (miRNAs) have gained considerable attention as potentially predictive biomarkers for a number of disorders. Mounting evidence suggests that miRNAs are differentially expressed in a variety of cancers and thus have potential for early detection, prognosis, and monitoring of response to treatment (Calin et al. 2006). Because of their small size and low concentrations, miRNAs in biological fluids are demanding targets for detection, requiring advanced diagnostic technologies. In this application note, protocols are described that allow for the specific detection of picomolar concentrations of cancer related miRNAs by utilizing the BiOptix 404Pi™ in combination with a signal enhancing monoclonal antibody that recognizes DNA-RNA hybrid duplexes (Hu et al. 2006).

Materials and Methods

Reagents
Oligonucleotides and synthetic microRNAs were obtained from Integrated DNA Technologies. Biotinylated anti-sense DNA oligos (sequences shown below) were used as a capture probes for detection.

Channel 1 Biot-AS miD-21 5′- /5BiosG/TC TGA TCA ACA TCA GTC TGA TAA GCT A -3′
Channel 2 Biot-AS miD-24 5′- /5BiosG/TC TGA CTG TTC CTG CTG AAC TGA GCC A -3′
Channel 3 Biot-AS miD-205 5′- /5BiosG/TC TGA CAG ACT CCG GTG GAA TGA AGG A -3′
Channel 4 Biot-AS miD-221 5′- /5BiosG/TC TCT GAA ACC CAG CAG ACA ATG TAG CT -3′

FirstChoice™ Human Liver Total RNA was purchased from Ambion (AM7960). The S9.6 mouse monoclonal antibody was generously provided by Dr. S. Leppla (NIH). All other reagents were obtained from Sigma-Aldrich.

Immobilization of DNA Probes BiOptix SA-150™ (Streptavidin) SensorChips were rinsed and inserted into the instrument. Individual biotinylated oligos (Biot-AS miD-24-1, Biot-AS miD-155, Biot-AS miD-205 and Biot-AS miD-221, 100 nM in PBS) were manually injected into channels 1-4, respectively. Loading was allowed to proceed for a total of 45 min with a second injection being performed at 20 min to maximize surface loading. At the end of the loading, each channel was rinsed with 2 mL of PBS before removing the SensorChip from the instrument.

 

General Assay Protocol
After inserting the charged SensorChip into the BiOptix 404Pi™, RNAse free HBS-Mg running buffer (10 mM HEPES, 150 mM NaCl, 15 mM MgCl2) was introduced until the baseline stabilized (~30 min). Various solutions of miRNA analogs (diluted in running buffer) were then injected at a flow rate of 60μL/min for 20 min followed by a 5 min dissociation phase with running buffer only. At the end of each measurement the sensor surface was regenerated using a 20 sec injection of 50 mM NaOH.

Experiments and Results

Specific Detection of miRNAs
A BiOptix 404Pi™ SensorChip previously loaded with four anti-sense capture DNA oligos was inserted into the BiOptix 404Pi™. Four separate samples of miRNA analogs (miR-21, 24, 205 and 221) were injected into the instrument to determine the specificity of the capture oligos. As expected, only the individual channel that contained the complementary oligonucleotide sequence showed a response (Figure 1). Minimal cross reactivity for the non-complementary oligos was observed with the various miRNAs.

Figure 1. Specific Detection of four MiRNAs. Variations in the total responses for complementary oligos are likely due to the different hybridization rates for duplex formation and the Tm’s for the oligos.

Multiplex Detection of miRNAs in total RNA extract

Using a SensorChip loaded as described above, the following three injections were performed:
1) 1 μg/mL total RNA extract from liver, 2) a mixture of miRNA analogs (21, 24, 205 and 221) at 100 nM each and 3) the same mixture spiked with 1 mg/mL total liver RNA extract. The total RNA extract alone showed a minimal background signal of ~10 RUs. As shown in Figure 2 the total RNA extract did not significantly interfere with the multiplex detection of a mixture of miRNA analogs.

Download the Full Application Note for Complete Results, Data and Figures