RollFISH achieves robust quantification of single-molecule RNA biomarkers in paraffin-embedded tumor tissue samples

Abstract

Single-molecule RNA fluorescence in situ hybridization (smFISH) represents a promising approach to quantify the expression of clinically useful biomarkers in tumor samples. However, routine application of smFISH to formalin-fixed, paraffin-embedded (FFPE) samples is challenging due to the low signal intensity and high background noise. Here we present RollFISH, a method combining the specificity of smFISH with the signal boosting of rolling circle amplification. We apply RollFISH to quantify widely used breast cancer biomarkers in cell lines and FFPE samples. Thanks to the high signal-to-noise ratio, we can visualize selected biomarkers at low magnification (20 × ) across entire tissue sections, and thus assess their spatial heterogeneity. Lastly, we apply RollFISH to quantify HER2 mRNA in 150 samples on a single tissue microarray, achieving a sensitivity and specificity of detection of HER2-positive samples of ~90%. RollFISH is a robust method for quantifying the expression and intratumor heterogeneity of biomarkers in FFPE tissues.

Single molecule chromogenic in situ hybridization assay for RNA visualization in fixed cells and tissues

“…However, when we increased the number of padlock probes to three, the RCPs that could be detected per cell also increased. Although the median detected HER2 signal spots in A549 for smCISH was around half of that of the rollFISH and smFISH (12 per cell for smCISH versus 21 per cell for rollFISH and 26 per cell for smFISH), we detected more HER2 signals in the MCF-7 cells (rollFISH detected more HER2 signals in A549 than in MCF-7 in the referenced paper [Wu et al 2018]). Considering that we were not using exactly the same cells as in the reference, we can conclude that our smCISH can achieve comparable detection efficiency as smFISH and rollFISH when using multiple padlock probes.…”

“…S5; Supplemental Table S1). When we compared our results to those of smFISH and rollFISH in Wu et al (2018), the detection efficiency of smCISH that used only one padlock probe was much lower than that of smFISH and rollFISH (Supplemental Table S1). However, when we increased the number of padlock probes to three, the RCPs that could be detected per cell also increased.…”

Evaluation of direct grafting strategies in Expansion Microscopy

“…In turn, this allows the use of signal amplification approaches in expansion microscopy, and the study of low-abundance species with high signal-to-noise ratios relative to standard approaches. Again, sequential or combined/orthogonal coupling of the multivalent linker to oligo tiling probes with hybridization chain reaction initiator sequence (HCR) [15][16][17][18] or amplification primers (RollFISH) [19][20][21] allows the creation of multifunctional ISH probe sets, in only a few hours and from commercial oligonucleotides. To demonstrate this, we conjugated tiling probe libraries to the smHCR initiator probes, all coupled through multifunctional linkers (Figure 4, panel a).…”

Direct detection of mRNA expression in microbial cells by fluorescencein situhybridization using RNase H-assisted rolling circle amplification

“…However, this method is time-consuming and confounded by background noise presumably due to nonspecific hybridization of the probe. Most currently available FISH methods, including RNAscope 12 and RollFISH 13 , rely on simple hybridization of oligonucleotide probes to mRNA sequences. Therefore, these methods cannot clearly distinguish between fluorescent signals originating from mRNA and genomic DNA, especially in microbes.…”