The Helicos® Digital Gene Expression (DGE) application enables direct cDNA-based gene expression profiling – providing you with a highly sensitive and specific transcriptome-wide view of the cell. This tool takes gene expression analysis closer to the biology of the cell.
The digital nature of the Helicos DGE application means:
- A simple hypothesis-free approach — no need to identify and characterize genes prior to conducting experiments
- Absolute and digital data that is capable of merging with data sets from other expression profiling technologies
- Performance and dynamic range comparable to qPCR
- Freedom from bias due to hybridization, ligation and amplification
- Obtaining cDNA-based counts for every gene in your sample and comparing expression levels across different genes, multiple samples, experiments, time points and platforms
- Quantifying global gene expression levels, detecting low-expressing genes and discovering novel expression events – all at a cost point lower than those of competitive approaches.
The Advantages
The Helicos® Genetic Analysis Platform allows direct digital transcript profiling. Directly measuring first-strand cDNA avoids the potential biases introduced through ligation or amplification techniques. Sample preparation is simple, scalable and cost-effective. Using direct cDNA sequencing to count gene transcripts also frees you from the constraints of hybridization biases and probe design. You can discover novel expression events and even analyze the transcriptomes of organisms for which arrays are not available.
The application of the Helicos® Genetic Analysis System to cDNA-based DGE analyses has been described in recent publications:
Quantification of the yeast transcriptome by single-molecule sequencing. Nat Biotechnol 27, 652-658.
Aberrant overexpression of satellite repeats in pancreatic and other epithelial cancers. Science, 331: 593-6.
Protocol dependence of sequencing-based gene expression measurements. PLoS One. 2011 May 6;6(5):e19287.
RNA sequencing and quantitation using the Helicos Genetic Analysis System. Methods Mol Bio. 2011;733:37-49.
Amplification-free digital gene expression profiling from minute cell quantities. Nat Methods. 2010;7 :619-21.
The Evidence: Sensitivity and Dynamic Range Superior to qPCR
With over eight million direct sequence reads per flow cell channel, the Helicos Digital Gene Expression application offers the sensitivity to detect rare transcripts (down to three transcripts per million) and a dynamic range comparable to or exceeding that of qPCR (greater than four orders of magnitude) – making Helicos Digital Gene Expression the simplest and most sensitive solution for comprehensive gene expression analysis. And because the approach is simple and unbiased, results are consistent and reliable (see graphs).
Figure 1, Left Panel. Intra-run technical replicates (different channels, same run) with a correlation coefficient of 0.999882, and R2 of 0.99978, 8.5 million reads vs. 6.5 million reads. The plot suggests a robust technique with good sensitivity. Measured expression levels in the mixtures show excellent agreement with the expected values over the entire dynamic range.
Figure 1, Right Panel. Inter-run technical replicates (different runs, same sample) with a correlation coefficient of 0.998094, and R2 of 0.996445, 15 million reads vs. 8 million reads.
The Approach
The Helicos Digital Gene Expression application uses a direct and simple approach to count mRNA molecules across the whole transcriptome and provide you with the truest representation of transcript abundance possible.
Figure 2. Helicos DGE Sample Preparation Methodology.
Using a total or PolyA RNA sample, mRNA is converted to first-strand cDNA with a poly-U primer and reverse transcriptase to generate one single cDNA molecule per poly-A transcript. This first-strand cDNA is then tailed without ligation, blocked on the 3’ end, and sequenced directly on the HeliScope™ Single Molecule Sequencer. The Sequencer produces hundreds of millions of individual reads, which are rapidly aligned to a reference database to count transcript expression levels for each gene.
Helicos Digital Gene Expression – Affordable and quantitative global gene expression profiling. Evaluate the expression of all your genes without the biases inherent to amplification.