To evaluate the reliability of the DASL assay for obtaining gene expression profiles (GEPs) from both low RNA input samples and CTCs, experiments were run with low predefined numbers of breast cancer cells (MCF7 and MDA-MB-468), spiked into a HDB at final concentrations of 400, 200, and 100 cells/5 mL.
Total RNA from captured cells was split in 2 equal aliquots and processed in duplicate on 2 distinct chips of the DASL platform together with total RNA (0.5-100 ng) isolated from the same in vitro cultured cells, in the same amplification/hybridization session.
 Nonstandard abbreviations: PT, primaiy tumor; CTC, circulating tumor cells; EpCAM, epithelial cell adhesion molecule; DASL, cDNA-mediated annealing, selection, extension, and ligation; HDB, healthy donor blood; IRCCS, Istituto di Ricovero e Cura a Carattere Scientifico; GEP, gene expression profile; Ct, cycle threshold.
Similar studies by Abramovitiz and Ton  had shown that a threshold [C.sub.t] value of [less than or equal to] 29 would signify sufficient RNA quality to give reproducible results in the cDNA-mediated annealing, selection, extension, and ligation (DASL) assay; this threshold was used in our study.
The whole genome DASL assay developed by Illumina is highly specialized to detect 1.3- to 2-fold changes in intact and partially degraded RNA from FFPE samples .
The raw DASL array data was exported via genomeStudio version 1.0 (Illumina, Inc.) to JMP Genomics (SAS Institute, Inc., Cary, NC, USA) for quantile normalization, principal component analysis (PCA), and log transformation.
Based in East London, registered charity DASL helps communities and individuals tackle the problems caused by drug and alcohol misuse through education, raising awareness and therapeutic services.
Michael O'Dwyer, DASL Newham Community Drug & Alcohol Team Senior Manager, explains: “DASL works alongside a range of external organisations, including health and social care providers.
We recognise that data security is going to be on the agenda for many organisations in 2014, particularly when it involves vulnerable individuals, so it is great to see charities such as DASL taking proactive steps to secure the information they share.
For each sample, 200 ng of total RNA was converted to cDNA and processed in the DASL assay as described previously (11,13).
1, the DASL assay gave highly reproducible intensity measurements for FFPE samples preserved for 1.5-3 years.
In a separate study, we used the DASL assay to monitor expression of 510 prostate cancer-related genes in >140 cancer and benign prostate hyperplasia FFPE samples.