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J Biomol Tech. 2010 September; 21(3 Suppl): S43.
PMCID: PMC2918210

Capture and Analysis of Newly Synthesized RNA A Novel Enabling Technology to Study High Resolution Gene Expression

H. Bharmal, S. Clarke, A. Lemire, and B. Agnew
Life Technologies Inc., Eugene, OR, United States

Abstract

RP-70

Newly transcribed RNA contains valuable information of the transcriptome. Development of enabling technologies using affinity nucleosides will help accelerate research that depends on high resolution gene expression. We describe an enabling tool based on click chemistry, which begins with metabolic incorporation of an ethynyl uridine (EU) into newly synthesized RNA. The purified total EU-RNA is reacted with biotin-azide using copper catalyzed click chemistry followed by binding to streptavidin beads. In this manner, the nascent RNA is captured for subsequent analysis by RT-qPCR, array analysis and sequencing. Toxic effects of EU were investigated using several cell health assays. Global transcriptome changes were monitored using microarray analysis. Our results show that nascent RNA can be captured and analyzed via RTqPCR and RNA-seq. Our results also indicate that no toxic effects on cell health are seen with treatments of 250 uM EU for over 24 hours. RTqPCR and microarray analysis also indicate that EU treatment does not alter the global transcription levels. Incorporation of EU for varying time periods followed by capture of nascent RNA and RTqPCR analyses showed changes in the nascent RNA copy number. Up until now there have been few methods to capture and characterize newly transcribed RNA. Traditional methods such as nuclear run on assays are cumbersome and do not reflect true physiology. RTqPCR has been used to gain information on the steady state RNA levels. The main limitation of all these approaches is that alterations in mRNA transcript (turnover) that strongly govern the patterns of expressed genes cannot be studied. Using the Click nascent RNA capture approach, for the first time we show RNA turnover rates for different mRNA transcripts and how they correlate with relative abundance of that transcript.


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