Keeping an Eye on the Messenger

Fluorescence-Based Tools to Image, Isolate, and Quantify RNA

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The importance of studying RNA cannot be overstated: not only is RNA the central intermediate between the genetic material and the protein machinery of the cell, but it also plays essential roles in regulating gene expression and serves as the genome for many viruses.

Fluorescence-based assays have been critical for the study of many of these processes. Molecular Probes® Click-iT® RNA assays enable the imaging of both cellular and viral RNA synthesis without the use of antibodies or radioactivity, and this same technology can be exploited to capture nascent RNA for subsequent analysis of specific transcripts. The Qubit® RNA assays, designed for use with our benchtop Qubit® 2.0 Fluorometer, offer highly sensitive fluorescence-based quantitation of RNA even in the presence of DNA, free nucleotides, and protein. In addition to the fluorescence-based tools discussed here, Life Technologies offers a comprehensive suite of reagents and instruments for all aspects of RNA research.

Imaging RNA Synthesis

Imaging newly transcribed (nascent) RNA in cells can provide powerful insights into the regulation of RNA synthesis and its transport within the cell. The Click-iT® RNA Imaging and HCS Kits allow for the detection of nascent RNA, both spatially and temporally, by fluorescence microscopy or high-content screening (HCS). These Click-iT® RNA assays employ the alkyne-modified nucleoside EU (5-ethynyl uridine), which is fed to cells, incorporated into RNA during transcription, and detected with an intensely fluorescent Alexa Fluor® azide via a “click” reaction between the alkyne and azide

The Click-iT® RNA assays provide an important advantage over traditional antibody-based BrU or BrUTP assays: the small size of the Alexa Fluor® azide detection reagent relative to the anti-BrU antibody enables more effective detection of both single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA). These assays are ideal for multiplex analyses that require the simultaneous imaging of nascent RNA and specific proteins of interest (Figure 1).

The Click-iT® RNA Alexa Fluor® Imaging Kits have been used in a number of recent studies to characterize both host and viral transcription in infected cells [1,2]. The levels of viral RNA synthesis and the subcellular localization of viral RNA can be measured using Click-iT® RNA assays after host transcription is inhibited (Figure 2).

 

Multiplex imaging with Click-iT® RNA assays 
Figure 1. Multiplex imaging with Click-iT® RNA assays. NIH/3T3 cells were incubated with 1 mM EU, formaldehyde-fixed, and permeabilized with Triton® X-100. EU incorporated into newly synthesized RNA (red) was detected using the Click-iT® RNA Alexa Fluor® 594 Imaging Kit and can be seen in the nucleus of several cells. Tubulin (green) was detected with anti–bovine α-tubulin antibody (mouse monoclonal 236-10501) and visualized with Alexa Fluor® 488 goat anti–mouse IgG antibody. Nuclei (blue) were stained with Hoechst® 33342.

 

Imaging coronavirus RNA synthesis 
Figure 2. Imaging coronavirus RNA synthesis. Murine LR7 fibroblast cells infected with mouse hepatitis virus (MHV) were fed with EU either at 4.15 hr or at 8.15 hr post-infection (pi). The Click-iT® RNA Alexa Fluor® 594 Imaging Kit was used to detect nascent RNA synthesis (red). Detection of double-stranded RNA (dsRNA) intermediates with antibodies (green) is also shown. The images demonstrate colocalization of newly synthesized RNA with dsRNA only at the early time point, indicating that the dsRNA viral intermediates are no longer functioning in RNA synthesis at the later time point. Figure reproduced with permission from the Journal of Virology [1] and Cornelis A.M. de Haan, Utrecht University, Utrecht, the Netherlands.

Capturing Newly Synthesized RNA

Because traditional gene expression profiling is based on the quantitation of total or steady-state mRNA, it is biased toward the detection of abundant, long-lived transcripts, which can obscure short-term changes in gene expression. A very different view of gene expression is captured with a snapshot of the newly transcribed RNA, traditionally accomplished with cumbersome nuclear run-on assays.

The Click-iT® Nascent RNA Capture Kit provides a simple, nonradioactive alternative to partitioning newly transcribed RNA from pre-existing RNA, thereby facilitating studies of RNA stability, RNA synthesis, transcriptional regulation, and RNA decay. This kit enables RNA—synthesized during a time window defined by administration of EU—to be selectively biotinylated via click coupling of the incorporated EU to biotin azide. The biotinylated RNA is then captured using streptavidin-functionalized magnetic beads; it can be used for reverse transcription and subsequent analysis by DNA sequencing, PCR, or microarray hybridization. Importantly, EU has no toxic effects at the concentrations used in the assay, and it has not been observed to alter global transcription [3].

For many transcripts, the measured change in relative gene expression is dramatically enhanced when nascent RNA is used for the analysis rather than total RNA (Figure 3). Indeed, a recent study demonstrated that deletion of the TFIIH kinase subunit Mat1 had little effect on the majority of steady-state mRNAs as measured by microarray analysis; however, a dramatic reduction in nascent transcripts—isolated using the Click-iT® Nascent RNA Capture Kit—was observed in the deletion mutant [4].

 

High-resolution gene expression analysis with the Click-iT® Nascent RNA Capture Kit 
Figure 3. High-resolution gene expression analysis with the Click-iT® Nascent RNA Capture Kit. Relative expression of the indicated genes was compared—with and without a treatment thought to affect apoptosis—using the Click-iT® Nascent RNA Capture Kit. For many transcripts, the measured change in relative gene expression (∆∆Ct) was dramatically enhanced when nascent RNA was used for the analysis rather than total RNA.

Quantitating RNA in the Presence of DNA and Protein

Accurate and sensitive nucleic acid quantitation is often critical for downstream analysis of RNA. Historically, DNA and RNA have been quantitated using spectrophotometry to measure absorbance at 260 nm. Although this method is commonly used, it cannot accurately distinguish between DNA, RNA, and protein. In addition, measurements are easily affected by other contaminants (e.g., free nucleotides, salts, and organic compounds) and variations in base composition. Furthermore, the sensitivity of spectrophotometry is often inadequate, prohibiting quantitation of DNA and RNA at low concentrations.

Fluorescence-based quantitation of nucleic acid is not only more sensitive but often more specific for the nucleic acid of interest.The Qubit® 2.0 Fluorometer is our second-generation benchtop fluorometer designed to work seamlessly with the Qubit® DNA, RNA, and protein assays. Together they form the Qubit® Fluorometric Quantitation Platform, an efficient combination of highly sensitive fluorescence-based quantitation assays and a pioneering benchtop instrument capable of unprecedented accuracy. The Qubit® Quantitation Platform provides a more selective, sensitive, and accurate method for quantitating nucleic acids than UV absorbance measurements, including those obtained with the NanoDrop® ND-1000 Spectrophotometer.

The Qubit® Quantitation Platform enables accurate measurement of both DNA and RNA from the same sample using a combination of kits. The Qubit® RNA assay has a linear detection range of 25–500 ng/mL (in the assay tube) and is selective for RNA even in the presence of an equal mass of DNA (Figure 4). In addition, we offer the Qubit® RNA Broad-Range (BR) Assay Kit, which provides a linear detection range of 0.1–5 µg/mL (in the assay tube). Qubit® Assay Kits are also available for the sensitive and specific quantitation of double-stranded DNA, single-stranded DNA, and protein.

Our similar Quant-iT™ RNA Assay Kit and Quant-iT™ RNA BR Assay Kit are designed for higher-throughput microplate readers, and each provides sufficient reagents for 1,000 assays.

 

Selectivity and sensitivity of the Qubit® and Quant-iT™ RNA assays 
Figure 4. Selectivity and sensitivity of the Qubit® and Quant-iT™ RNA assays. The Qubit® and Quant-iT™ RNA assays have a linear detection range of 25–500 ng/mL (in the assay tube or well) and are selective for RNA, even in the presence of an equal concentration of DNA. Triplicate 10 µL samples of E. coli rRNA, DNA, or a 1:1 mixture of RNA and DNA were assayed with the Quant-iT™ RNA Assay Kit. Fluorescence was measured at 630/680 nm and plotted against the concentration of the RNA alone, the DNA alone, or the RNA component in the 1:1 mixture. The inset is an enlargement of the graph to show the sensitivity of the assay for RNA. Background fluorescence has not been subtracted.

Essentials From the RNA Experts

In addition to the fluorescence-based tools discussed here, Life Technologies offers a comprehensive portfolio of reagents, instruments, and custom services for all aspects of your RNA research—from RNA extraction and RNA stability to RNAi, qPCR/transcriptome analysis, and ncRNA analysis. This expansive suite of products features the trusted Ambion® TRIzol®, PureLink®, MagMAX™, mirVana™, and Dynabeads® RNA technologies that offer the high yield and quality demanded by your research.

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