Figure 1.
Experimental design of the LASH assay.
(A) Schematic representation of the ligase-assisted sandwich hybridization assay (LASH assay) for detection of non-labeled miRNA. A target miRNA hybridizes to complimentary D-probe and C-probe, resulting in a tertiary complex. Both C-probe and D-probe are capped with a phosphate group at the 5′ end and a hydroxyl group at the 3′ end; this permits T4 DNA ligase to ligate the 5′ end of C-probe to the 3′ end of D-probe, and to ligate the 5′ end of D-probe to the 3′ end of the target miRNA. (B) A bright-field image of droplets spotted on a coverslip by an ink-jet machine. Scale bar, 400 µm. (C) Fluorescence images of 6-FAM-labeled C-probe (left) immobilized on a substrate and Alexa647-labeled D-probe (right) bound to C-probe in the presence of 1.25 nM miR-143. Scale bar, 100 µm.
Figure 2.
Optimization of D-probe complementary sequence length.
(A) Complementary sequences of four probe sets for miR-143. (B) The fluorescence intensities of D-probes bound to C-probes in the presence of T4 DNA ligase. Data represent the mean ± S.E. (n = 3). (C) Correlation between input miR-143 and the signal of D-probe-143-(8) in the presence (filled circle) or absence (open circle) of T4 DNA ligase. The upper axis indicates the final concentration of input miR-143 in the hybridization chamber while the lower axis indicates the total amount of input miR-143. Data in the linear range were fitted by a linear expression (gray lines). Data represent the mean ± S.E. (n = 3).
Figure 3.
Distinction between homologous miRNAs in the LASH assay.
(A) Schematic drawing of two-color detection experiments. Each D-probe can hybridize and be ligated to C-probe-141/200a-(14) in the presence of each target miRNA. (B) Complementary sequences of probe sets for miR-141 and miR-200a. Two nucleotides of the miR-200a sequence are different from those of miR-141 (red characters). These two nucleotides are located in the sequence that binds to the D-probe. On the other hand, the underlined sequences at the 5′ side of both miRNAs are identical and can bind to the common complimentary C-probe-141/200a-(14). The percentages of cross-hybridization of miR-141 and miR-200a to the opposite D-probes were evaluated.
Figure 4.
Quantification of extracellular miR-143 in total miRNA released from HEK293 cells using qRT-PCR or the LASH assay.
The transfection of a primary miR-143 vector promoted the exocytosis of miR-143 from HEK293 cells compared with that from wild-type HEK293 cells, as described previously [28]. The LASH assay achieved the same quantification of miR-143 in total miRNA as qRT-PCR.
Figure 5.
Quantification of miRNAs in total RNA samples derived from two types of human blood.
The amounts of miRNAs in 1 µg of total RNA were estimated by qRT-PCR or LASH assay. The expression profiles of the two blood samples were reproducible between the two methods. Data represent the mean ± S.E. (n = 3).