- Centrifuge the lyophilized oligonucleotides at 600gfor
2 min and carefully add TE to the tube after centrifuging to
dissolve the probes to 100μM with vigorous shaking. - SYBR Green I must be protected from light and stored at 4C.
The concentration of SYBR Green I stock solution is 10,000
and the optimal final concentration is 4. Dilute directly from
stock solution before use. - Target probe (Target oligo) consists of two regions that linked
with a ‘TTTTT’ structure. The first region located on the 3^0 -
end can hybridize with capture probe in the 96-well plate, and
the second region on the 5^0 -end triggers the cascade chain
reaction of two hairpin sets. - Make sure the wells are completely sealed so the solution in the
well won’t evaporate during incubation. - Unlike previously reported HCR systems in solution, our assay
is an on-site amplification technology. Leaky hairpins will be
washed off without contributing to the background. Thus, we
can maximize polymerization without being overly concerned
about leakiness by using shorter hairpins, improving the sensi-
tivity and making hairpin design easier. - Mix the hairpin probes just before use. Probe X1* and probe
X2 are paired with final concentration of 1μM. Cascade chain
reaction will be triggered in the presence of target probe. - Hairpin probe X1* has an additional hangout sequence at the
50 -end that does not form double strand structure after poly-
merization, but instead, act as the initiating probe for the
second step of polymerization. - The direct RNA detection assay is comprised of two parts: the
first part is a specific RNA capture, and the second part is signal
amplification through 2-dimensional hybridization chain reac-
tions. Multiple LE probes per target could be a first step to
signal amplification by providing many initial probes for the
subsequent detection. Adaptor probes that simultaneously
hybridize to the tails of LEs mediate the capture and amplifica-
tion systems. The introduction of adaptor probe enables inde-
pendent designing of DNA probes in capture stage and
detection stage, making the assay more flexible. As a result,
the method can be used to detect any suitable RNA or DNA
targets by designing new target-specific CEs and LEs probes,
while keeping the same hairpin sets used in this study. Also, a
sequence of “TTTTT” is inserted into the two binding regions
of adaptor probe, resulting in better signal amplification than
that without “TTTTT”. - Multiple oligonucleotide probes capture the RNA on 96-well
plate in the capture stage. Based on our previous study, this
Hybridization Chain Reaction for Direct RNA Detection 195