Journal: bioRxiv

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

doi: 10.1101/2021.11.28.470233

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 (18S 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 analysed 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 hours. 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. 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 ( Mueller et al ., 2013 ; Tsanov et al ., 2016 ).

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