Bead-Enriched Catalyzed Hairpin Assembly for the Flow Cytometric Detection of microRNA via FRET signal readout

The accurate quantification of microRNAs (miRNAs) is of great significance for the various biomedical applications. Herein, a facile bead-enriched catalyzed hairpin assembly (CHA) assay for the flow cytometric miRNA analysis...

USA).The total RNA extracted from cells was subject to the standard procedures of the beadenriched CHA for miRNA analysis procedures stated in the main text to quantitatively evaluate the level of let-7a.
Quantification of let-7a in the total RNA by RT-PCR method.The stem-loop reverse-transcription PCR (RT-PCR) protocol is referred to the methods in the literatures with some modifications.Reverse transcription reaction: The reverse transcription reaction was carried out in the mixture with 1 µL target miRNA (or total small RNA sample), 1.2 µL of RNase-free water, 1 µL of 5 × RT buffer (50 mM Tris-HCl, 75 mM KCl, 3 mM MgCl 2 ), 1 µL   4. Detection results of let-7a with the proposed bead-enriched CHA assay by using 1.2 × 10 5 beads.
To evaluate the analytical performance of the proposed bead-enriched CHA assay, approximate 1.2 × 10 5 beads are employed to analyze the concentration of let-7a.As shown in Figure S2, the FRET signal continuously rises with the concentration of let-7a increasing from 50 pM to 300 pM.Notably, as low as 50 pM let-7a-produced FRET signal can be well discriminated from the blank signal.

Calculation of the detection limit
In the Table S2, we provide the average FRET signal and standard deviation of nine parallel test results of the blank control.According to the 3σ criterion and the slope of the calibration curve, the detection limit of let-7a is calculated to be 0.7 pM (Equation 1).
Moreover, from another angle, if we interrogate the detection limit value by using 2.5 pM target (whose FRET signal can be clearly discriminated from that of blank control) as a point on the calibration curve in the linear range, according to the Equation 2, the detection limit is calculated to be 0.65 pM.These results are consistent.

Let
of 2.5 mM dNTPs, 0.2 µL of 200 U/µL ProtoScrip II reverse transcriptase, 0.5 µL of 1 of 40 U/µL RNase inhibitor.The 5 µL mixture was treated with following conditions: 30 min at 16 °C, 30 min at 42 °C, 5 min at 85 °C and then held at 4 °C.Quantitative realtime PCR analysis: 5 µL transcription product was added into the PCR reaction mixture with a final volume of 10 µL.The PCR reaction mixture consists of 200 nM forward primer (5, and 1 × PCR buffer (10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl 2 , and 0.001(w/v) gelatin, pH 8.3).The 10 µL PCR reaction mixture was incubated in a StepOne Real-Time PCR System (Applied Biosystems, USA) according to the following thermal cycling conditions: hot start at 95 °C for 2 min, followed by 50 cycles of 95 °C for 15 s, and 2. Nucleic acid sequences used in this work.

3.
Figure S1.Schematic illustration of the detailed principle of miRNA-initiated CHA based on the toehold-mediated strand displacement.

Figure S2 .
Figure S2.Fluorescence intensity of Cy3 vs. fluorescence intensity of FAM scattering plots of the beads in the presence of different concentration of let-7a by using 1.2 × 10 5 beads.

Figure S3 .
Figure S3.Fluorescence intensity of Cy3 vs. fluorescence intensity of FAM scattering plots of the beads in the presence of 0 and 2.5 pM of let-7a.

Comparison of different nucleic acid detection methods based on CHATable S3 .
Comparison of different nucleic acid detection methods based on CHA