Strength in numbers: quantitative single‐molecule RNA detection assays

Abstract

Gene expression is a fundamental process that underlies development, homeostasis, and behavior of organisms. The fact that it relies on nucleic acid intermediates, which can specifically interact with complementary probes, provides an excellent opportunity for studying the multiple steps—transcription, RNA processing, transport, translation, degradation, and so forth—through which gene function manifests. Over the past three decades, the toolbox of nucleic acid science has expanded tremendously, making high‐precision in situ detection of DNA and RNA possible. This has revealed that many—probably the vast majority of—transcripts are distributed within the cytoplasm or the nucleus in a nonrandom fashion. With the development of microscopy techniques we have learned not only about the qualitative localization of these molecules but also about their absolute numbers with great precision. Single‐molecule techniques for nucleic acid detection have been transforming our views of biology with elementary power: cells are not average members of their population but are highly distinct individuals with greatly and suddenly changing gene expression, and this behavior of theirs can be measured, modeled, and thus predicted and, finally, comprehended. WIREs Dev Biol 2015, 4:135–150. doi: 10.1002/wdev.170For further resources related to this article, please visit the WIREs website.Conflict of interest: The authors have declared no conflicts of interest for this article.

Temporally coordinated signals progressively pattern the anteroposterior and dorsoventral body axes

“…In situ transcript detection methods are qualitative and describe a transcript domain spatially, but do not provide quantitative data of transcript levels. However, recent advances in quantitative, single-molecule RNA transcript detection offer new possibilities [240–242]. For example, there are reports that bmp ligand expression during gastrulation is graded across the ventral half embryo, with highest expression ventrally and lowest expression laterally [144,178], while others do not report a difference in bmp expression [127,128,130,137,243].…”

Simultaneous Single-Cell In Situ Analysis of Human Adenovirus Type 5 DNA and mRNA Expression Patterns in Lytic and Persistent Infection

“…PLP-based RCA methods have also been applied to detect HAdV-5 E1A alternatively spliced mRNAs ex situ (28) and porcine circovirus type 2 virus DNA in situ (29). Whereas the usage of PLPs for mRNA detection has received a standard molecular technique status already (30), simultaneous and multiplexed analyses of DNA and mRNAs across various biological specimens have not been reported.…”

Fixed and live visualization of RNAs in Drosophila oocytes and embryos

“…A full understanding of dynamic processes like mRNA localization requires the ability to visualize RNA molecules in live cells, in real time. Numerous methods have been developed to this end, including injection of in vitro synthesized fluorescently labeled transcripts and the application of conditionally fluorescent RNA-binding probes like molecular beacons, RNA aptamers, and RNA intercalating dyes (see Gaspar and Ephrussi for detailed review [17]). While these reagents can be readily delivered to cultured cells, introducing them into Drosophila oocytes and embryos is problematic, requiring microinjection or inefficient and potentially harmful permeabilization schemes.…”

“…While these reagents can be readily delivered to cultured cells, introducing them into Drosophila oocytes and embryos is problematic, requiring microinjection or inefficient and potentially harmful permeabilization schemes. In contrast, genetically encoded fluorescent tagging methods based on the high affinity interaction of bacteriophage proteins with cognate RNA stem-loops [17] are particularly well suited for Drosophila given the ease of transgenesis.…”