Single Cell Analysis of MiR-155 and MiR-146a Regulation of the Macrophage Immune Response using In Situ Hybridization Chain Reaction
Author:
Nikita SinhaMentor:
David Baltimore, Robert A. Millikan Professor of Biology, California Institute of TechnologyMicroRNAs play a significant role in the complex gene regulatory network that tightly regulates mammalian hematopoiesis to ensure balanced and appropriate hematopoietic output. Here we develop a technique to detect target miRNAs on a single-cell level by in situ hybridization chain reaction (HCR), in which RNA probes complementary to mRNA targets trigger chain reactions where fluorophore-labeled RNA hairpins self-assemble into tethered fluorescent amplification polymers. While theoretically identifying and imaging miRNAs by HCR in situ hybridization should be no different from mRNAs, the short sequence and low endogenous signal intensity of miRNAs required us to optimize the technique. Our results show that the protocol for miRNAs is different from that of mRNAs in that cells need to be treated with proteinase K to make miRNAs available for probe binding and that the cells need to be fixed with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide to prevent leakage of miRNAs out of the cell during the hybridization and amplification steps. Next we aim to understand the fine-tuning of NF-kB signaling at the single-cell level in the context of miR-155 and miR-146a. This in turn will elucidate how microRNA expression can confer different functional properties to subsets of macrophages and how this might contribute to pathological inflammatory responses.