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Preparation of long single-strand DNA concatemers for high-level fluorescence in situ hybridization

ABSTRACT: Fluorescence in situ hybridization (FISH) is a powerful tool to visualize transcripts in fixed cells and tissues. Despite the recent advances in FISH detection methods, it remains challenging to achieve high-level FISH imaging with a simple workflow. Here, we introduce a method to prepare long single-strand DNA concatemers (lssDNAc) through a controllable rolling-circle amplification (CRCA). Prepared lssDNAcs are used to develop AmpFISH workflows. In addition, we present its applications in different scenarios. AmpFISH shows the following advantages: 1) enhanced FISH signal-to-noise ratio (SNR) up to 160-fold compared with single-molecule FISH; 2) simultaneous detection of FISH signals and fluorescent proteins or immunofluorescence (IF) in tissues; 3) simple workflows; and 4) cost-efficiency. In brief, AmpFISH provides convenient and versatile tools for sensitive RNA/DNA detection and to gain useful information on cellular molecules using simple workflows. Cao et al. present a method to prepare long single-strand DNA concatemers (lssDNAc) through a controllable rolling-circle amplification (CRCA), used to develop AmpFISH workflows. Their method is suitable for labelling to both individual mRNA molecules and chromosomes and can also be multiplexed with immunofluorescence, allowing for sensitive RNA/DNA detection.

SUBMITTER: Cao D

PROVIDER: S-EPMC8545947 | biostudies-literature |

REPOSITORIES: biostudies-literature

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