Multiplex gene expression profiling of CTC populations – performance

HyZip technology offers outstanding sensitivity, specificity and reproducibility, which are all critical for the molecular analysis of rare CTCs.

Sensitivity: Detects a single cancer cell or 3 transcript molecules. Sensitivity is driven by:

  • No need to split samples – multiplex amplification in a single-tube reaction
  • No RNA extraction required minimizing nucleic acid losses. Direct hybrid capture from lysis buffer.
  • Flow-through micro-array provides enhanced speed and sensitivity due to the increased surface area available for binding and the elimination of diffusion rate as a limiting factor.
Varying amounts of non-human control mRNA were processed with HyZip - three replicates of each mRNA amount (black) and of diluent-only samples (green). As few as 3 molecules of target RNA were detected, although the response to very few molecules is subject to stochastic variability. The response was approximately linear (slope c.1.0).

Reproducibility:

  • No global pre-amplification required, reducing the potential for amplification bias
  • Direct hybrid capture from lysis buffer – avoiding any extraction anomalies

Donor blood from each of 3 healthy normal volunteers spiked with CaOV3 cells and processed with Parsortix platform. The harvests were analyzed with HyZip Women’s Health Panel. Each plot represents the overlay of 4 individual repeats for each donor. The median CV for all genes was 28.0%, representing the variability of the entire process including spiking, cell harvesting and the HyZip process.

Specificity:

  • Multiple recognition events (two target primers to amplify the sequence and one target probe to detect the amplified product)
  • Detection of multiple genes limits false negative results
Blood from healthy normal volunteers was either unspiked or spiked with a single CaOV3 cell and processed on a Parsortix cartridge. Lysates of each Parsortix harvest were analyzed with the HyZip technology. The data demonstrates that the gene expression profile of a single spiked cell can be determined against a background of several thousand blood cells.

To discuss how you might incorporate gene expression profiling of CTCs into your research studies or clinical trials contact us at EUsales@angleplc.com or NAsales@angleplc.com