Search bioRxiv⌕ Search

Biology subjects

Kohman, R. E.

Publications and source records attributed to Kohman, R. E..

2 recordsLinked to original sources
bioRxiv2018

Barcoded oligonucleotides ligated on RNA amplified for multiplex and parallel in-situ analyses

We present Barcoded Oligonucleotides Ligated On RNA Amplified for Multiplexed and parallel In-Situ analysis (BOLORAMIS), a reverse-transcription (RT)-free method for spatially-resolved, targeted, in-situ RNA identification of single or multiple targets. For this proof of concept, we have profiled 154 distinct coding and small non-coding transcripts ranging in sizes 18 nucleotides in length and upwards, from over 200, 000 individual human induced pluripotent stem cells (iPSC) and demonstrated compatibility with multiplexed detection, enabled by fluorescent in-situ sequencing. We use BOLORAMIS data to identify differences in spatial localization and cell-to-cell expression heterogeneity. Our results demonstrate BOLORAMIS to be a generalizable toolset for targeted, in-situ detection of coding and small non-coding RNA for single or multiplexed applications.

cell biology↗
bioRxiv2018

Multiplexed imaging using same species primary antibodies with signal amplification

Immunofluorescence (IF) imaging using antibodies to visualize specific biomolecules is a widely used technique in both biological and clinical laboratories. Standard IF imaging methods using primary antibodies followed by secondary antibodies have low multiplexing capability due to limited availability of primary antibodies raised in different animal species. Here, we used a DNA-based signal amplification method, Hybridization Chain Reaction (HCR), to replace secondary antibodies to achieve multiplexed imaging using primary antibodies of the same species with superior signal intensity. To enable imaging with DNA-conjugated antibodies, we developed a new antibody staining protocol to minimize nonspecific binding of antibodies caused by conjugated DNA oligonucleotides. We also expanded the HCR hairpin pool from previously published 5 to 13 for highly multiplexed in situ imaging. We finally demonstrated multiplexed in situ protein imaging using the technique in both cultured cells and mouse retina sections.

cell biology↗