Journal: ACS synthetic biology

Article Title: Nucleic Acid Strand Displacement with Synthetic mRNA Inputs in Living Mammalian Cells

doi: 10.1021/acssynbio.8b00288

Figure Lengend Snippet: a) Domain-level schematic of SDprobe_v1: Individual functional domains are annotated with their respective primary features in parentheses. Half-arrows indicate 3’-ends of the corresponding strands. The fluorophore and quencher are denoted by red and black circles respectively. b) Schematic of probe interaction with the 3’-UTR target sites (blue domains). Probes bind to the target sites via the short toehold domain, which initiates a 3-way branch migration step, ultimately displacing the quencher-labeled strand and thereby unquenching the fluorophore. Co-localization of multiple fluorophores on a single mRNA significantly increases the signal. c) Experimental workflow for detection of mRNAs using strand displacement probes: Probes are delivered via electroporation to cultured cells. Cells are incubated for 1 hour at 37°C allowing the probes to interact with mRNA target sites. Co-localization of up to 96 probes per mRNA generates a bright fluorescent spot detectable with fluorescence microscopy. After incubation, sequential image frames are collected at every 0.3 um z-distance across the cell height and the acquired frames are combined to generate maximum intensity projections representing the entire cell volume. These images are processed using the FISH-Quant33 MATLAB tool; a filtering step performs background signal correction and spots are pre-detected based on a threshold signal cut-off. The pre-detected spots are then fitted and examined for accuracy followed by spot quantification, where the spots are either selected or rejected based on signal intensity and elimination of false-positives. The selected spots represent the number of detected mRNAs per cell.

Article Snippet: These images are processed using the FISH-Quant 33 MATLAB tool; a filtering step performs background signal correction and spots are pre-detected based on a threshold signal cut-off.

Techniques: Functional Assay, Migration, Labeling, Electroporation, Cell Culture, Incubation, Fluorescence, Microscopy

Journal: eLife

Article Title: Global analysis of contact-dependent human-to-mouse intercellular mRNA and lncRNA transfer in cell culture

doi: 10.7554/eLife.83584

Figure Lengend Snippet: ( A ) Heatmap summarizing transfer of human mRNAs in Co-culture compared to Mix. qRT-PCR was performed on RNA samples from MBS-MEF Single culture, Mix, and Co-culture samples from two independent biological replicates and the presence of transferred RNA was detected with human-specific oligos for ten transferred genes, as identified in . Four less transferred genes were used as negative controls. Human β-actin was used as a positive control. The color of the heatmap corresponds to the row-wise Z-scores of Ct values of the indicated genes after normalizing for an internal control (18 S rRNA). Increasing darkness corresponds to increasing gene expression. All but one gene (i.e. GNAS) showed good agreement with the RNA-seq results and have a higher fold-change as compared to the Mix sample. Each box represents an average of three independent technical replicates. ( B–C) Verification by smFISH . Three genes (KRT8, PSAP, and CCND1) that demonstrated high level of transfer by RNA-Seq were chosen to be analyzed by smFISH. Acceptor cells (MBS-MEFs) were co-cultured with MCF7 cells together on fibronectin-coated coverslips at a ratio of 1:1 for 12 hr. Following co-culture, the cells were fixed and smFISH was performed using Quasar 570-labeled oligonucleotide probes complementary to the human-specific RNA of the indicated genes and Cy5-labeled probes specific for the MBS sequence. The transfer of mRNAs was detected by wide-field microscopy and quantified using a MATLAB program, FISH-Quant. ( B ) smFISH images. Representative smFISH images of MBS-MEF and MCF7 single cultures, and MCF7 cells in co-culture with MBS-MEFs. Labels: gray, Q570-labeled smFISH probes; blue, DAPI staining of the nucleus. Donor and acceptor cells were distinguished by the high expression of β-actin-MBS (identified by Cy5-MBS probes) in MBS-MEF cells only (not shown). Scale bar: 10 µm. ( C ) Quantification of the number of mRNAs of two independent experiments. The left panels show the number of mRNAs expressed for the indicated genes in the MCF7 cells only, while the right panel shows the number of corresponding mRNAs in MBS-MEF cells alone or in co-culture. Each dot represents the number of indicated mRNAs detected in a single cell, as measured by FISH-Quant. Horizontal red lines represent the average number of mRNAs. ** - p≤0.01; **** - p≤0.0001.

Article Snippet: The number of mRNAs (FISH spots) were scored using a MATLAB based GUI program, FISH-Quant (FQ), as described ( ; ).

Techniques: Co-Culture Assay, Quantitative RT-PCR, Positive Control, Control, Gene Expression, RNA Sequencing, Cell Culture, Labeling, Sequencing, Microscopy, Staining, Expressing