RNA Detection

sufficient for clinical applications. Linear, one-dimensional chain

reactions are adopted in most current HCR amplification methods.

Although branched HCR or other higher dimensional amplifica-

tion may presumably give much higher sensitivity, the challenges of

steric hindrance and the difficulties to form branched oligonucleo-

tides in solution still make it hard to realize.

Here, we demonstrate an improved HCR-based RNA detec-

tion method (Figs.3 and 4)[5]. By adopting a sandwich RNA

capturing assay, the nucleic acid extraction procedure is bypassed,

allowing high-throughput, ELISA-like sample processing in 96-

well plates. Simultaneously, a novel, onsite two-dimensional

branched HCR assembly is made possible by detecting target cap-

tured on solid support. The sensitivity and the specificity of RNA

detection were improved compared with the traditional linear HCR.

2 Materials

All oligonucleotides are purchased commercially and are PAGE-

purified. Ultrapure water and analytical grade reagents are used in

all runs. 5
SSC buffer (750 mM NaCl, 75 mM sodium citrate,

pH 7.4) is used for all hybridization reactions unless indicated

specifically.

2.1 Agarose Gel
Electrophoresis

1. Standard equipment of running agarose gels (electrophoresis
   tank, power supply, gel tray, and combs).

Target

X1

X1

X1

X2

X2

X2

Hybridize and wash

X2

X2

X2

The 2nd dimension HCR

Hybridize and wash

Target

X1

X1

X1

A1

A1

A2

A2

X1

X2

A1

A2

The 1st dimension HCR

... ...

...

...

...

Fig. 3Two-dimensional HCR on solid surface (seeNote 10). In the 2D HCR, The target probe hybridizes on its
30 -half with the capture probe conjugated on the solid surface; the 5^0 -half of the target then opens the hairpin
probe X1, triggering the cascade chain reaction of the hairpin set X to form a nicked double helix with extra
single-strand hangout branches. After washing, hairpin set A is added; the overhang branches from X1*
opened A1 and initiated the HCR of the hairpin set A, generating a 2D HCR product. SYBR Green I is employed
in the detection phase to generate fluorescent signals. We also detected the fluorescent signal generated by
hairpin sets with the same sequences but labeled with fluorescein isothiocyanate (FITC). It was observed that
unlabeled hairpin probes generated higher signal than labeled probes [5]. Figure adapted from [5] with
permission from Elsevier

Hybridization Chain Reaction for Direct RNA Detection 189