- Nano Cap Analysis of Gene Expression (nanoCAGE) is a refined adaptation of the CAGE technique, designed specifically for situations where the starting RNA quantity is extremely limited—such as single cells, small tissue biopsies, or rare cell populations.
- While conventional CAGE requires microgram quantities of RNA, nanoCAGE is optimized for nanogram amounts, enabling transcription start site (TSS) profiling and promoter usage analysis from scarce biological material. The method preserves the key principle of CAGE—capturing and sequencing the very 5′ end of capped transcripts—while integrating modifications to minimize sample loss and amplify the captured sequences without introducing significant bias.
- At the core of nanoCAGE is a strategy that combines the 5′-cap trapping principle with template-switching reverse transcription. In this approach, capped RNAs are reverse transcribed using a primer complementary to the poly(A) tail (for mRNAs) or other specific sequences. When the reverse transcriptase reaches the 5′ cap, it incorporates a few extra cytosines at the cDNA’s end due to its terminal transferase activity. A specially designed template-switching oligonucleotide (TSO), carrying a known sequence and an adapter, then anneals to these extra cytosines and serves as an extended template for the reverse transcriptase, allowing the incorporation of the adapter sequence directly at the 5′ end of the cDNA. This process effectively “captures” the capped transcript’s start site without needing separate cap-trapping steps, reducing sample handling and material loss.
- Once the cDNA with both 5′ and 3′ adapters is generated, it undergoes limited-cycle PCR amplification to increase material yield while minimizing over-representation of certain sequences. The amplified cDNA is then subjected to high-throughput sequencing, typically producing short reads (20–30 bp) corresponding to the TSS. These reads are aligned to the reference genome to identify active promoters, alternative transcription initiation events, and expression levels, just like in standard CAGE analysis. Because each read represents a transcript’s starting point, nanoCAGE provides quantitative as well as positional information about transcription initiation, even from extremely low-input samples.
- nanoCAGE has found significant applications in single-cell transcriptomics, early developmental biology, and rare cell type characterization, where input RNA is inherently scarce. It enables researchers to explore promoter usage heterogeneity, transcriptional regulation at fine resolution, and the dynamics of gene expression changes in systems previously inaccessible to standard genomic assays. The method has been used extensively in projects such as the single-cell FANTOM5 initiative, contributing to high-resolution promoter atlases.
- Despite its power, nanoCAGE requires meticulous optimization to prevent artifacts from template switching and to maintain strand specificity, but when properly implemented, it opens the door to detailed promoter and enhancer activity mapping from previously intractable sample types.
