A new gene expression technique adapted for single molecule sequencing has enabled researchers at the RIKEN Omics Science Center (OSC) to accurately and quantitatively measure gene expression levels using only 100 nanograms of total RNA. The technique, which pairs RIKEN’s Cap Analysis of Gene Expression (CAGE) protocol with the Helicos Genetic Analysis System developed by Helicos BioSciences Corporation, opens the door to the detailed analysis of gene expression networks and rare cell populations.
recent years, next-generation DNA sequencers have produced an
increasingly detailed picture of how genes are expressed at the
molecular level. The transcriptional output of these genes – the RNA
copies produced from DNA – has revealed a richness of complexity in
transcript structure and function, providing insights into the
molecular-level properties of cancers and other diseases.
One of the most powerful methods for analyzing RNA transcripts is the Cap Analysis of Gene Expression
(CAGE) protocol developed at the RIKEN OSC. A unique approach, CAGE
enables not only high-throughput gene expression profiling, but also
simultaneous identification of transcriptional start sites (TSS)
specific to each tissue, cell or condition.
HeliScopeCAGE, the OSC research team has adapted the existing CAGE
protocol for use with the revolutionary HeliScope Single Molecule
Sequencer. Unlike earlier sequencers, the HeliScope Sequencer does not
employ polymerase chain reaction (PCR) amplification to multiply a small
number of DNA strands for analysis, a process which can introduce
biases into data. Instead, the HeliScope Sequencer actually sequences
the DNA strand itself, enabling direct, high-precision measurement.
In a paper published in Genome Research, RIKEN researchers confirm that this direct approach reduces
biases and generates highly reproducible data from between 5 micrograms
to as little as 100 nanograms of total RNA. A comparison using a
leukemia cell line (THP-1) and a human cervical cancer cell line (HeLa)
further shows that results from the technique are closely correlated to
those from traditional microarray analysis. By making possible
high-precision gene expression analysis from tiny samples, HeliScopeCAGE
greatly expands the scope of research at the OSC, strengthening the
institute’s role in Japan as a hub for next-generation genome analysis.
The RIKEN Omics Science Center is developing a versatile analysis system called the “Life Science Accelerator (LSA)” with the objective of advancing omics research. LSA is a multi-purpose, large-scale analysis system that rapidly analyzes molecular networks. It collects various genome-wide data at high throughput from cells and other biological materials, comprehensively
analyzes experimental data, and thereby aims to elucidate the molecular
networks of the sample. The term “accelerator” was chosen to emphasize
the strong supporting role that this system will play in supporting and
accelerating life science research worldwide.
Mutsumi Kanamori-Katayama, Masayoshi Itoh, Hideya Kawaji, Timo
Lassmann, Shintaro Katayama, Miki Kojima, Nicolas Bertin, Ai Kaiho,
Noriko Ninomiya, Carsten O. Daub, Piero Carninci, Alistair R. R. Forrest
and Yoshihide Hayashizaki. Unamplified Cap Analysis of Gene Expression
on a single molecule sequencer. Genome Research (2011).