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Sample GSM1274525 Query DataSets for GSM1274525
Status Public on Dec 31, 2014
Title MCF10A_PCR_SC_R3
Sample type RNA
 
Source name MCF10A single cell
Organism Homo sapiens
Characteristics cell line: MCF10A
Growth protocol 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 single cell
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
City Manchester
ZIP/Postal code M20 4BX
Country United Kingdom
 
Platform ID GPL17989
Series (2)
GSE52714 Flexible multiplatform RNA profiling at the single cell level applied to enriched cancer initiating cells: RT-PCR
GSE52717 Flexible multiplatform RNA profiling at the single cell level applied to enriched cancer initiating cells

Data table header descriptions
ID_REF
VALUE Log2 Cq values

Data table
ID_REF VALUE
NAB35.102S 21.62639
NAB35.105S 18.94799
NAB35.106S 0
NAB35.108S 18.76863
NAB35.110S 23.83426
NAB35.111S 16.12267
NAB35.113S 21.72056
NAB35.114S 0
NAB35.116S 9.95479
NAB35.118S 17.57448
NAB35.11I 22.23735
NAB35.122S 0
NAB35.125S 18.258
NAB35.127S 0
NAB35.12S 24.33388
NAB35.131S 21.61726
NAB35.13I 20.5691
NAB35.14I 17.70312
NAB35.15I 0
NAB35.161S 17.0302

Total number of rows: 173

Table truncated, full table size 2 Kbytes.




Supplementary data files not provided
Processed data included within Sample table
Processed data are available on Series record

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