The human epithelial cell lines MCF7 and MCF10A were grown in DMEM (Gibco, Paisley, Scotland) supplemented with 10% heat-inactivated FCS, 2.5% HEPES buffer (pH 7.2), 0.1% β-mercaptoethanol and 2mM L-glutamine (all from Sigma, UK). Both cell lines were maintained in a 5% CO2 humidified incubator at 37°C and were routinely tested for the presence of mycoplasma.
Extracted molecule
total RNA
Extraction protocol
RNA (1ng-25pg) or cell lysates in CLS were adjusted to 6.75ml volume and RNA-amplified using the EpiStem RNA-Amp Kit according to the manufacturers protocols (EpiStem, Manchester, UK). Briefly the samples underwent oligo-dT priming and 5’ capping prior to x35 cycles of PCR amplification using the conditions 90ºC 30 sec, 42ºC 2 min and 72ºC 6 min. Following amplification all samples were purified using a MoBio UltraClean® PCR Clean-Up Kit (Carlsbad, CA, USA) and quantified on a NanoDrop spectrophotometer.
Label
sybr green
Label protocol
Gene expression was assessed using the SmartChip Real-Time PCR System (WaferGen BioSystems, Fremont, USA). Sample and assay mixes were prepared with SensiFASTTM SYBR Hi-ROX (Bioline) in 384-well source plates using a Freedom Evo 150 robot (Tecan). Assay and sample mixes were then automatically loaded into the nanowells of a MyDesign SmartChip with WaferGen’s MultiSample NanoDispenser using a ‘384 assays x 12 samples’ dispensing layout. In total 8 chips were prepared and analyzed, corresponding to a total of 96 samples for 384 unique targets. The final reaction volume per nanowell was 100 nl, with an equivalent of 100 pg unamplified cDNA (total RNA equivalents) loaded per reaction. SmartChips were run in the SmartChip Cycler, and the cycling conditions were comprised of 3 minutes activation at 95°C, and 40 cycles of 30 seconds at 95°C and 60s at 60°C, followed by a dissociation curve analysis from 60°C to 95°C. Cq values, generated by the software from the SmartChip Cycler were used for down-stream data-analysis.
Hybridization protocol
n/a
Scan protocol
n/a
Description
Amplified cDNA from 1ng RNA
Data processing
As an initial processing stage, 35-Cq was calculated for each probe to facilitate simple, intuitive analysis (it was a 35 cycle qPCR). A subset of informative probes were selected based on consistent expression above a background threshold of 10 in either cell line (mean (35-Cq)>10 and stdev<1.5 in either MCF7 or MCF10A cDNA generated from 1ng amplified RNA), excluding repetitive probes. A single probe per gene was then selected from this informative set based on visual inspection of the data for MCF7 and MCF10A unamplified cDNA and cDNA generated from 1ng amplified RNA.
Submission date
Nov 25, 2013
Last update date
Dec 31, 2014
Contact name
Ged Brady
Organization name
Caner Research UK Manchester Institute
Department
Clinical and Experimental Pharmacology
Street address
Cancer Research UK Manchester Institute, The University of Manchester, Wilmslow Rd
