Fig. 1

Implementation and validation of RollFISH. a Workflow of RollFISH in comparison to smFISH and standard RCA. Each ODN in a RollFISH probe consists of 30nt complementary to the target RNA sequence and of 46nt orthogonal to the human transcriptome, serving as docking sequence for a padlock probe. A hinge sequence of four thymidines (T) is included between the two sequences to facilitate recognition and binding of the padlock probe to the docking sequence. b Representative images of RollFISH, smFISH, and standard RCA for HER2 in A549 cells. The magnification used and the number of ODNs per probe is shown. Blue, Nuclei. Scale bar, 10 µm. c Quantification of the number of spots per cell in images of which those in b are representative. Boxes extend from the 25th to the 75th percentile. Whiskers extend from 2.5 to 97.5 percentiles. The line inside each box represents the median value. Three experiments were performed for each assay, and all the data were pooled into one box plot. n, total number of cells analyzed. d Comparison of HER2 levels detected in six different cell lines by RollFISH and RNA-seq. RollFISH was repeated twice and the mean value for each cell line is plotted