Analysis of newly transcribed, or nascent, RNA provides information that cannot be obtained from analysis using traditional total RNA preparations. The new Click-iT® Nascent RNA Capture Kit makes it simple to partition newly transcribed RNA from pre-existing RNA. After purification, the nascent RNA can be used for virtually any downstream application. The kit provides a streamlined, nonradioactive alternative to cumbersome nuclear run-on⁄run-off protocols. In addition, the procedure benefits from the specificity and stability of click chemistry that is missing from procedures based on bromine- and thio-modified ribonucleosides.
One Reaction, Myriad Possibilities
The Click-iT® Nascent RNA Capture Kit procedure is simple. First, ethynyl uridine (EU), which contains an alkyne group, is fed to cells or animals. A unique benefit of Click-iT® EU is that it is incorporated efficiently into RNA without any apparent effect on the global transcriptome. Once the EU is incorporated, RNA is isolated and a biotin azide is “clicked on” to the newly synthesized pool of EU-modified RNA. The biotinylated nascent RNA is then captured using streptavidin magnetic beads.
Nascent RNA obtained using the Click-iT® Nascent RNA Capture Kit has been validated for use in both TaqMan® Assay– and SYBR® Green dye–based qPCR, microarray analysis, and sequencing using the SOLiD™ System. Applications include analysis of gene expression, RNA stability, regulation of transcription; and rates of RNA synthesis, decay, and degradation.
Boost the Resolution of Your Gene Expression Analysis
Traditional gene expression profiling is based on the quantitation of total mRNA. It is biased to detect changes in short-lived transcripts, because the slow turnover of long-lived transcripts can obscure short-term changes in their synthesis. Figure 1 illustrates high-resolution gene expression analysis facilitated using the Click-iT® Nascent RNA Capture Kit method. The bars on the graph represent the change in relative gene expression (ΔΔCt) in response to an experimental treatment. For six of the nine transcripts analyzed, the fold difference in Ct values was greatly enhanced when nascent RNA was evaluated instead of total RNA.
Figure 1. High-resolution gene expression analysis with the Click-iT® Nascent RNA Capture Kit. Relative expression (ΔCt) of the indicated genes was compared with and without a treatment thought to affect apoptosis (ΔΔCt). For many transcripts, the change in relative gene expression measured was dramatically enhanced when nascent RNA was used for the analysis rather than total RNA.
Expand Your Research Without Expanding Your Budget
The kit includes reagents for 40 labeling and capture reactions that provide enough template to run ≥400 qPCR reactions. Priced at ~$1 per qPCR assay, the Click-iT® Nascent RNA Capture Kit provides an affordable way to capture nascent RNA, enabling novel transcriptome research.
Discovery, Not Just Recovery
Incorporate Click-iT® metabolic analogs to label newly synthesized DNA, RNA, protein, sugars, and posttranslational modifications. Learn more at Click Chemistry Solutions for Biological Research.