Abstract

Assessment of multiplexed quantitative ability of in situ hybridization chain reaction based upon fluorescent intensity and mRNA grain count correlation

Kumar V, Krolewski DM, Foltz M, Akil H, Watson SJ
52nd Annual Meeting of Society for Neuroscience. 2023.

The cost effectiveness of in situ hybridization chain reaction (HCR) compared to other multiplexing in situ hybridization approaches (e.g., RNAscope) makes it a desirable method for RNA expression analysis. However, variations in the individual probe binding and the extent of linear chain amplification, can influence both the fluorescence intensity and the mRNA grain (copy number) count. Such variations could become more prominent in mammalian brain tissues, particularly in the postmortem human brain tissues where disease conditions can alter the RNA integrity and thus the overall binding sensitivity of probes. Here, we assess the multiplexed quantitative ability of HCR for mRNA analysis in multiple cell types in fresh-frozen brain tissue, and explore key factors including the choice of fluorophores, tissue preparation, experimental conditions, and imaging parameters which critically determine the overall sensitivity and effectiveness of the HCR quantitation. Using multi-round HCR, we analyze Vglut1, Gad2, Fgf12 transcripts based upon fluorescence intensity and grain count estimation. These genes under basal conditions in rat brain, represent a dynamic range of expression pattern in terms of cell number and mRNA copies. Our preliminary tests for Fgf12 and Gad2 suggest a linear association between voxel intensity and grain counts for hairpin concentration range 2-30 nM and incubation times 1-2 hrs. Thereafter, increasing the number of probe pairs, longer hairpin incubation times and/or higher concentrations appears to yield brighter and larger spots which fail to resolve properly, thus, inversely effects the segmentation of individual grains particularly in high grain density (subcellular) locations. We further explore and highlight the key factors which determine consistency, sensitivity, and optimum signal to noise ratio, essential for both qualitative and quantitative analysis. Based upon our observations, choice of fluorophore (AlexaFluor-647 or -546 vs. AF-488) hairpin conjugates, tissue fixation (fresh frozen vs. perfused) and the abundance of transcript level (grain density and cell number) critically determines the effectiveness of the HCR for accurate grain counting with respect to the mRNA expression. For example, Gad2 in fresh-frozen tissues and Npy when detected with AF-647 or -546 consistently showed biologically accurate cell counts in comparison to perfused tissues using AF-488 or -405, respectively. Similarly, genes with high density of grains such as Sst present challenge to accurately resolve individual grains and count despite using low concentration of hairpins (0.6nM to 1.25nM) or fewer probe pairs (1-2 pairs).