Project description:We developed an amplified single-molecule RNA FISH method called clampFISH 2.0. To validate its performance, we used clampFISH 2.0 on two cell lines, WM989 A6-G3 cells and WM989 A6-G3 cells grown in vemurafenib, and across 10 genes, compared clampFISH 2.0 spot counts to the FPKM values from bulk RNA sequencing.
Project description:We present Barcoded Oligonucleotides Ligated On RNA Amplified for Multiplexed and parallel In Situ analyses (BOLORAMIS), a reverse transcription-free method for spatially-resolved, targeted, in situ RNA identification of single or multiple targets. BOLORAMIS was demonstrated on a range of cell types and human cerebral organoids. Singleplex experiments to detect coding and non-coding RNAs in human iPSCs showed a stem-cell signature pattern. Specificity of BOLORAMIS was found to be 92% as illustrated by a clear distinction between human and mouse housekeeping genes in a co-culture system, as well as by recapitulation of subcellular localization of lncRNA MALAT1. Sensitivity of BOLORAMIS was quantified by comparing with single molecule FISH experiments and found to be 11%, 12% and 35% for GAPDH, TFRC and POL2RA respectively. To demonstrate BOLORAMIS for multiplexed gene analysis, we targeted 96 mRNAs within a co-culture of iNGN neurons and HMC3 human microglia cells. We used fluorescence in situ sequencing to detect error-robust 8-base barcodes associated with each of these genes. We then used this data to uncover the spatial relationship among cells and transcripts by performing single-cell clustering and gene-gene proximity analyses. We anticipate the BOLORAMIS technology for in situ RNA detection to find applications in basic and translational research.