Fig. 1.

Hybridization chain reaction increases detection of GC rich repeats over FISH. a FISH probe with a 5′ conjugated fluorophore. Signal strength is dependent on number of RNA molecules present. b In R-HCR, two probe types are used: the initiator probe which binds directly to the RNA of interest, and 5' fluorophore conjugated hairpin probes (H1 and H2) complementary to 3′ and 5′ extensions on the initiator probe. Upon binding, H1 and H2 unfold to reveal new binding sites for the other hairpin probe. In this way, signal from one RNA molecule is amplified > 100 fold, dramatically enhancing detection. c, d MEFs transfected with (G₄C₂)₇₀-NL-3xFlag (c) or CGG₁₀₀-3xFlag (d) vectors and probed with indicated concentrations of either FISH or R-HCR probe. e MEFs transfected with (G₄C₂)₇₀-NL-3xFlag expressing vector (top) or CGG₁₀₀-3xFlag vector (bottom) and treated with DNase (left) or RNase (right) prior to R-HCR. f ICC-R-HCR of MEFs transfected with antisense (G₂C₄)₄₇-NL-3xFlag (top) or (CCG)₆₀-NL-3xFlag (bottom) expressing plasmids. Top: N = 434; Bottom: N = 38.Error bars indicate 95% confidence intervals (CI). Scale bar = 20 μm in c, d, e; 50 μm in f