In comparison with other analysis methods

• What are the benefits of CAGE compared to microarry?
  • Offers highly accurate and detailed gene expression analysis by targeting transcription start site (TSS) rather than whole genes. It was estimated that there are approximately 50,000 genes in human genomes, but, over 185,000 TSSs have been identified. CAGE is capable of recovering most of these TSSs.
  • Quantification based on each TSS instead of each gene provides a chance to find out genes differently expressed that was not detected by microarray.
  • Does not require DNA chips loaded with probes, allowing the analysis of novel genes.
  • Provides a wider dynamic range to allow analysis of both high- and low-expressing genes.
  • Enables the detection of enhancer RNA (eRNA) that is generally bi-directionally expressed in a low level.
  • Precise identification of TSS enables a better prediction of Transcription Factor Binding motifs than RNA-seq.

• What are the disadvantages of CAGE compared to microarry?
  • The turnaround time for a CAGE project takes around 1.5-2 months
  • Publicly available analysis tools are not sufficient (We will provide support in data analysis in request).
  • The amount of RNA (few µg) required as the starting material for analysis is somewhat larger than microarray.

• What are the benefits of CAGE compared to RNA-seq?
  • Offers highly accurate and detailed gene expression analysis by targeting transcription start site (TSS) rather than whole genes. It was estimated that there are approximately 50,000 genes in human genomes, but, over 185,000 TSSs have been identified. CAGE is capable of recovering most of these TSSs.
  • Quantification based on each TSS instead of each gene provides a chance to find out genes differently expressed that was not detected by RNA-seq.
  • Provides a wider dynamic range to allow analysis of both high- and low-expressing genes.
  • The PCR-free library preparation process decreases the sequence-dependent bias and improve the quantification accuracy.
  • Enables the detection of enhancer RNA (eRNA) that is generally bi-directionally expressed in a low level.
  • Precise identification of TSS enables a better prediction of Transcription Factor Binding motifs than other methods.

• What are the disadvantages of CAGE compared to RNA-seq?
  • Not suitable for detection of transcript splicing-variant and genetic variation such as SNP.
  • Suitable only for analysis of species with a known reference genome, such as human, mouse, rat, nematode, zebrafish, Arabidopsis thaliana, etc.
  • Publicly available analysis tool is insufficient. (We can provide technical support in data analysis).
  • The amount of RNA (few µg) required as the starting material for analysis is somewhat larger than RNA-seq.

• Is it compatible with ChlP-seq or Methylation analysis?
  • Binding sites of transcription factors (TFs) and DNA methylation sites can be determined by ChIP-seq and methylation analysis, respectively, but the binding of TFs or DNA methylation are not always coupled with the transcription activity. CAGE is a reliable method for monitoring transcription activity from TSSs, thus providing a verification of results of ChIP-seq and/or methylation analysis.

About RNA Samples

• What are the sample requirements?
  • A minimum of 6μg (CAGE library preparation service; 12μg is preferable) or 5μg (CAGE kit) total RNA with RIN above 7.0.
  • The recommended concentrations are 1µg/µl. In case that concentration is lower than 100ng/µL, we will concentrate it using SpeedVac before we start library preparation.
  • The solvent should be RNase-Free Water.
  • We recommend RNA extraction using QIAGEN’s extraction kit, RNeasy, but RNA extractions by using other similar kit are also acceptable.
  • In case you have to perform EtOH precipitation, please do not use glycogen as the precipitation carrier.

• How about when it is difficult to provide 6µg of total RNA?
  • We can perform CAGE analysis from a minimum of 0.1µg RNA samples, but PCR will be necessary.

• How should I send my total RNA samples?
  • Package the samples with dry ice when sending.

About CAGE

• Is CAGE reliable in gene expression measurement?
  • In principle, CAGE is more accurate than microarray or RNA-seq. CAGE can be taken as a variation of RNA-seq, specialized in sequencing only 5’end of cDNA. Because the library preparation process is completely PCR-free, an RNA molecule generates only one tag. Therefore, the tag number precisely represents the expression level of a gene. In comparison, the canonical RNA-seq sequenced the whole region and generates multiple tags from a single RNA molecule, the expression level of a specific gene has to be normalized by the length of RNA. Therefore, CAGE gets a more precise quantification of gene expression with a lower throughput of sequencing.

• What are CAGE’s unique features?
  • The most unique feature of CAGE is that it provides information on the expression level of transcripts from the TSS site on a 1bp resolution level. In human, it is estimated that there are about 50,000 genes generating both protein-coding and non-coding RNA, but over 185,000 Transcription Start Sites (TSS) have been identified, suggesting that there are at least 3 alternative promoters for each gene on average. Analysis of gene expression in a TSS level, rather than in a whole gene level, provides a much more sensitive and extensive view of gene regulation. CAGE satisfies the requirement of TSS-based analysis of gene expression. Besides, FANTOM project have analyzed over 1,000 sample for human and 300 samples for mouse by CAGE, researchers on human or mouse can evaluate one’s CAGE result by comparing it to FANTOM data.
  • Unlike RNA-seq, CAGE is PCR-free and therefore more immune to sequence-dependent bias.
  • One disadvantage is the transcript Splicing-variant and sequence variation such as SNP can not be analyzed, as it only reads 5’ ends of an RNA.

• What is Cap structure?
  • A guanosine methylated on the 7 position connected to 5’ end of mRNA via a 5 to 5 triphosphate linkage. Capping is a kind of post-transcriptional RNA processing which functions on the protection of the 5’ end of RNA.

• What kinds of institutions are using CAGE?
  • CAGE is used in academic institutions on fields such as medicine, dentistry, veterinary medicine, pharmacology, agriculture and marine studies. CAGE is also used in the pharmaceutical industry and biotechnology industry.

• Is it possible to do CAGE data analysis by myself?
  • Several analysis softwares have been developed specialized for CAGE data analysis.

• Can you do pathway analysis or Gene Ontology analysis?
  • Gene Ontology analysis is included in our basic analysis service. You can also conduct pathway analysis and Gene Ontology analysis by yourself using we btools such as DAVID, GSEA and PANTHER.

• How long does a CAGE project take?
  • The turnaround time for a CAGE project is between 1.5-2 months. The entire process consists of 2-4 weeks for library preparation, 3-8 weeks for library prep + sequencing and 4-8 weeks for library prep + seq + analysis, respectively.

• Are there papers utilizing CAGE analysis?
  • Several dozens of papers have been published. Multiple papers have been published in Nature and there are sure to be more in the future. The papers primarily come from RIKEN, but there are also articles from other institutions. (see a reference list)

• What did the Riken FANTOM project do with CAGE?
  • The Riken FANTOM project analyzed approximately 1000 kinds of samples using the CAGE method. With those results, the activity of approximately 185,000 promoter sites and 44,000 enhancer sites were identified and we discovered that many gene control sites exhibit behavior that is specific to each cell. Half of the identified promoters were discovered for the first time. Although the enhancers were already known, it was the first time such a substantial amount of activity related to enhancers was analyzed with a large number of samples.