SLIC-CAGE is a novel development of the cap-trapper CAGE protocol which can generate complex high-quality libraries for as little as 5-10 nanograms of cellular RNA.
Cap analysis of gene expression (CAGE) is a technique for quantitative genome-wide mapping of transcriptome start sites in addition to 5’end-centred expression profiling of RNA polymerase II transcripts. The technique can be used to identify core promoter sequences across a genome and has been used in a number of high profile research projects including ENCODE, modENCODE, FANTOM3 and FAMTOM5.
The classic CAGE protocol employs a cap-trapper step to select mature mRNA transcripts and subsequently pull down tagged RNA:cDNA hybrids using streptavidin beads. This step of the protocol necessitates the use of a high amount of starting cellular RNA (30-50 μg), which limits its use on scarce biological samples. Improvements to the original CAGE protocol (nAnT-iCAGE) reduced this requirement down to circa 5 μg of cellular RNA, but remains prohibitive for many samples. A related but distinct technique, nanoCAGE, eliminates the cap-trapper step and can be employed with 50-500 ng of cellular RNA, but produces lower quality libraries compared to CAGE protocols with retain the cap-trapper step.
The SLIC-CAGE protocol uses a specially designed, selectively degradable carrier RNA to supplement the sample RNA. The carrier RNA protects the sample RNA from losses due to non specific binding on beads or tubes during the protocol. Homing endonuclease recognition sites are embedded in the carrier RNA, allowing it to be removed from the sample prior to amplification and sequencing. This approach allows SLIC-CAGE to produce libraries of comparable complexity and quality to the current gold standard CAGE methodology (nAnT-iCAGE) with 1000-fold higher sensitivity.
SLIC-CAGE represents a demonstrable improvement in sensitivity over current gold standard CAGE protocols. SLIC-CAGE is likely to find widespread application in transcriptome mapping as it expands the applicability of the technique to scarce samples.
- Provides quantitative, genome wide mapping of transcription start sites.
- Produces complex libraries comparable to gold standard CAGE methodologies.
- Demonstrates a 1000 fold improvement in sensitivity over current methodologies, producing libraries from as little as 5-10 ng of cellular RNA.
The invention is covered by a patent application (PCT ref.