• 1 Department of Genetics, Harvard Medical School, Boston, MA 02115, USA
  • 2 Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA
  • 3 McGovern Institute, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
  • 4 Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA
  • 5 Media Arts and Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
Nucleic Acids Res, 2021 06 04;49(10):e58.
PMID: 33693773 DOI: 10.1093/nar/gkab120


We present barcoded oligonucleotides ligated on RNA amplified for multiplexed and parallel insitu 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 POLR2A, respectively. To demonstrate BOLORAMIS for multiplexed gene analysis, we targeted 96 mRNAs within a co-culture of iNGN neurons and HMC3 human microglial 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.

* Title and MeSH Headings from MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.