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 cloned libraries derived from small cellular RNAs.
The ExactSTART™
Small RNA Cloning Kit* generates a cloned library derived from small RNA transcripts, starting with a total RNA sample. The kit selectively tags and converts small RNAs that contain 5´-monophosphate/ 3´-hydroxyl ends (5´ pN—OH 3´) or 5´-triphosphate/3´-hydroxyl ends (5´ pppN—OH 3´) to amplified dsDNA. Species of RNA tagged by the kit include miRNA, other small noncoding RNAs, and small primary transcripts. The amplified dsDNA is ligated into the pCDC-1K™ Cloning-Ready Vector (provided in the kit) and then transformed into a competent E. coli host (supplied by the user).
An optimized RNA ligation reaction is used to tag the 5´ end, and cDNA synthesis is used to tag the 3´ end of the RNAs (Fig. 1). 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 and subsequently cloned.
Note: Plant miRNAs cannot be cloned with this kit, due to the 2´-O-methyl group at the 3´ end of the RNA.
Benefits
- Strong selection process for cloning of miRNAs and other small noncoding RNAs and primary transcripts with 5´-monophosphorylated or 5´-triphosphorylated ends (Table 1).
- Fast reactions: Clone and transform cells in 2 days.
- Includes pCDC-1K™ Cloning-Ready Vector (linearized and dephosphorylated).
| Table 1. Identification of characterized human miRNAs in a HeLa cDNA library prepared using the ExactSTART™ Small RNA Cloning Kit. Ninety-six randomly selected clones from the library were analyzed by sequencing. |
|
Figure 1 (click to enlarge). Schematic overview of the process for the ExactSTART™ Small RNA Cloning Kit. |
| hsa-let-7a |
1 |
| hsa-let-7i |
1 |
| hsa-miR-15b |
1 |
| hsa-miR-18a |
1 |
| hsa-miR-19b |
2 |
| hsa-miR-20a |
2 |
| hsa-miR-21 |
7 |
| hsa-miR-23a |
1 |
| hsa-miR-24 |
2 |
| hsa-miR-26a |
1 |
|
|
| hsa-miR-27b |
1 |
| 1 hsa-miR-92a |
7 |
| hsa-miR-96 |
1 |
| hsa-miR-99a |
1 |
| hsa-miR-125a-5p |
1 |
| hsa-miR-181a |
1 |
| hsa-miR-196a |
1 |
| hsa-miR-221 |
1 |
| hsa-miR-365 |
1 |
| hsa-miR-484 |
1 |
|
|
*Patent pending.
ExactSTART™ End-Tagged Double-Strand cDNA Synthesis Kit for Small RNA
ExactSTART™ Eukaryotic mRNA 5´- & 3´-RACE Kit
ExactSTART™ Full-Length cDNA Library Cloning Kit
RNA 5´ Polyphosphatase
TransforMax™ EC100™ Electrocompetent E. coli
TransforMax™ EC100™ Chemically Competent E. coli
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