Dev Growth Differ 2025 Aug 13;676:336-343. doi: 10.1111/dgd.70018.

Universal Improvement of In Situ Hybridization Chain Reaction by Reducing Background Signals Caused by Single Probes.

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The in situ hybridization chain reaction (HCR) method involves designing multiple target sequences and a pair of split probes for each target. One split probe contains the complementary sequence for half of the target along with part of the initiation sequence. The other split probe contains the complementary sequence for the remaining half of the target sequence and the rest of the initiation sequence. The complete initiation sequence composed of both probes is capable of initiating a chain reaction of hairpin DNAs. This theoretical mechanism minimizes the background signal caused by a single probe; however, very low background signals have been observed in experiments. While these weak signals are not a significant problem in many cases, they can interfere with experiments if the expression of the target gene is very low, making the background signal noticeable. Reducing such background signals would benefit many scientists working with diverse species and sample types. To address this issue, we hypothesized that a single probe could bind and open the hairpin DNA through partial complementary sequences, acting as a bridge between hairpin DNA and samples through nonspecific binding. Our findings show that the addition of random oligonucleotides during the pre-hybridization and hybridization steps reduced background signals by approximately 3-90 times. This simple and easy modification of the in situ HCR technique improves the signal-to-noise ratio and facilitates the detection of mRNAs with very low expression levels.

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