Gene Expression Analysis |
Transcriptome Discovery
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Transcriptome Analysis: ExactSTART™ Tools for Rapid and Efficient cDNA Preparation for Deep Sequencing, Cloning, and Expression Profiling (940K PDF poster)
Applications
Generation of dsDNA from small RNAs for:
- High-throughput (next-gen) sequencing templates.
- Small RNA RT-PCR templates.
- Labeled target for small RNA microarray analyses.
- Small RNA libraries for cloning.
The ExactSTART™ End-Tagged Double-Strand cDNA Synthesis Kit for Small RNA* selectively tags and converts small RNAs that contain 5´-monophosphate and 3´-hydroxyl ends (5´ pN—OH 3´), or 5´-triphosphate and 3´-hydroxyl ends (5´ pppN—OH 3´) to amplified dsDNA. Species of RNA tagged by the kit include mammalian miRNA, other small noncoding RNAs, and small primary transcripts.
An optimized RNA ligation reaction is used to tag the 5´ end, and a high-efficiency polyadenylation/cDNA synthesis reaction is used to tag the 3´ end of the RNAs (Fig. 1). Intervening size-separation by gel electrophoresis is not required. An optional step, using a unique RNA 5´ Polyphosphatase* enzyme (discovered and characterized by EPICENTRE scientists) enables tagging and dsDNA synthesis of small primary transcripts with 5´-triphosphorylated ends. An initial, simple RNA size-selection procedure removes large RNAs such as mRNA, and 18S and 28S rRNA, to ensure that only small RNAs are converted to dsDNA.
Benefits
- Strong selection process yields libraries with a very high proportion of dsDNA derived from small (<200-base) RNAs with 5´-monophosphorylated or 5´-triphosphorylated ends.
- Rapid and simple RNA size-selection procedure without the need for columns or gel electrophoresis.
- One-day protocol.
- User-defined PCR primers can be used to generate dsDNA for a specific downstream application (Fig. 1).
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Figure 1 (click to enlarge). Schematic overview of the process for the ExactSTART™ End-Tagged Double-Strand cDNA Synthesis Kit for Small RNA. |
*Patent pending
ExactSTART™ Eukaryotic mRNA 5´- & 3´-RACE Kit
ExactSTART™ Full-Length cDNA Library Cloning Kit
ExactSTART™ Small RNA Cloning Kit
RNA 5´ Polyphosphatase
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