Barcoded oligonucleotides ligated on RNA amplified for multiplexed and parallel in situ analyses.

Autor:	Liu S; Department of Genetics, Harvard Medical School, Boston, MA 02115, USA., Punthambaker S; Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.; Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA., Iyer EPR; Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.; Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA., Ferrante T; Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA., Goodwin D; McGovern Institute, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.; Media Arts and Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA., Fürth D; Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA., Pawlowski AC; Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.; Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA., Jindal K; Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.; Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA., Tam JM; Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.; Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA., Mifflin L; Department of Genetics, Harvard Medical School, Boston, MA 02115, USA., Alon S; McGovern Institute, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.; Media Arts and Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA., Sinha A; McGovern Institute, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.; Media Arts and Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.; Harvard-MIT Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA., Wassie AT; McGovern Institute, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.; Media Arts and Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02142, USA., Chen F; Media Arts and Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.; Broad Institute, Massachusetts Institute of Technology, Cambridge, MA 02142, USA., Cheng A; Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA., Willocq V; Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA., Meyer K; Department of Genetics, Harvard Medical School, Boston, MA 02115, USA., Ling KH; Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.; Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia., Camplisson CK; Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.; Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA., Kohman RE; Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.; Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA., Aach J; Department of Genetics, Harvard Medical School, Boston, MA 02115, USA., Lee JH; Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA., Yankner BA; Department of Genetics, Harvard Medical School, Boston, MA 02115, USA., Boyden ES; McGovern Institute, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.; Media Arts and Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02142, USA.; Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02142, USA.; Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USAHoward Hughes Medical Institute, Chevy Chase, MD 20815, USA., Church GM; Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.; Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA.
Jazyk:	angličtina
Zdroj:	Nucleic acids research [Nucleic Acids Res] 2021 Jun 04; Vol. 49 (10), pp. e58.
DOI:	10.1093/nar/gkab120
Abstrakt:	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. (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)
Databáze:	MEDLINE
Externí odkaz:	Zobrazit plný text záznamu