title: Compaction of rolling circle amplification products increases signal integrity and signal-to-noise ratio.
abstract: Rolling circle amplification (RCA) for generation of distinct fluorescent signals in situ relies upon the self-collapsing properties of single-stranded DNA in commonly used RCA-based methods. By introducing a cross-hybridizing DNA oligonucleotide during rolling circle amplification, we demonstrate that the fluorophore-labeled RCA products (RCPs) become smaller. The reduced size of RCPs increases the local concentration of fluorophores and as a result, the signal intensity increases together with the signal-to-noise ratio. Furthermore, we have found that RCPs sometimes tend to disintegrate and may be recorded as several RCPs, a trait that is prevented with our cross-hybridizing DNA oligonucleotide. These effects generated by compaction of RCPs improve accuracy of visual as well as automated in situ analysis for RCA based methods, such as proximity ligation assays (PLA) and padlock probes.
authors.affiliation: Department of Immunology, Genetics and Pathology, Uppsala University, Science for Life Laboratory, Biomedical center, SE-75108 Uppsala, Sweden.
authors.forename: Carl-Magnus
authors.initials: CM
authors.lastname: Clausson
authors.affiliation: Department of Immunology, Genetics and Pathology, Uppsala University, Science for Life Laboratory, Biomedical center, SE-75108 Uppsala, Sweden.
authors.forename: Linda
authors.initials: L
authors.lastname: Arngården
authors.affiliation: Department of Information Technology, Centre for Image Analysis, Uppsala University, Science for Life Laboratory, SE-75105 Uppsala, Sweden.
authors.forename: Omer
authors.initials: O
authors.lastname: Ishaq
authors.affiliation: Department of Immunology, Genetics and Pathology, Uppsala University, Science for Life Laboratory, Biomedical center, SE-75108 Uppsala, Sweden.
authors.forename: Axel
authors.initials: A
authors.lastname: Klaesson
authors.affiliation: Department of Immunology, Genetics and Pathology, Uppsala University, Science for Life Laboratory, Biomedical center, SE-75108 Uppsala, Sweden.
authors.forename: Malte
authors.initials: M
authors.lastname: Kühnemund
authors.affiliation: Department of Immunology, Genetics and Pathology, Uppsala University, Science for Life Laboratory, Biomedical center, SE-75108 Uppsala, Sweden.
authors.forename: Karin
authors.initials: K
authors.lastname: Grannas
authors.affiliation: Department of Immunology, Genetics and Pathology, Uppsala University, Science for Life Laboratory, Biomedical center, SE-75108 Uppsala, Sweden.
authors.forename: Björn
authors.initials: B
authors.lastname: Koos
authors.affiliation: Department of Biochemistry and biophysics, Stockholm University, Science for Life Laboratory, Svante Arrhenius väg 16C, SE-10691 Stockholm, Sweden.
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Qian
authors.affiliation: Department of Information Technology, Centre for Image Analysis, Uppsala University, Science for Life Laboratory, SE-75105 Uppsala, Sweden.
authors.forename: Petter
authors.initials: P
authors.lastname: Ranefall
authors.affiliation: Department of Biochemistry and biophysics, Stockholm University, Science for Life Laboratory, Svante Arrhenius väg 16C, SE-10691 Stockholm, Sweden.
authors.forename: Tomasz
authors.initials: T
authors.lastname: Krzywkowski
authors.affiliation: Science for Life Laboratory, Royal Institute of Technology, Box 1031, SE-171 21 Solna, Sweden.
authors.forename: Hjalmar
authors.initials: H
authors.lastname: Brismar
authors.affiliation: 1] Department of Immunology, Genetics and Pathology, Uppsala University, Science for Life Laboratory, Biomedical center, SE-75108 Uppsala, Sweden [2] Department of Biochemistry and biophysics, Stockholm University, Science for Life Laboratory, Svante Arrhenius väg 16C, SE-10691 Stockholm, Sweden.
authors.forename: Mats
authors.initials: M
authors.lastname: Nilsson
authors.affiliation: Department of Information Technology, Centre for Image Analysis, Uppsala University, Science for Life Laboratory, SE-75105 Uppsala, Sweden.
authors.forename: Carolina
authors.initials: C
authors.lastname: Wählby
authors.affiliation: Department of Immunology, Genetics and Pathology, Uppsala University, Science for Life Laboratory, Biomedical center, SE-75108 Uppsala, Sweden.
authors.forename: Ola
authors.initials: O
authors.lastname: Söderberg
mesh_terms: D004270:DNA, Circular / Q000737:chemistry* / Q000235:genetics*; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000235:genetics*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*; D059629:Signal-To-Noise Ratio
chemical_list: D004270:DNA, Circular; D004277:DNA, Single-Stranded