See growth protocol. These ES cell lines were maintained normally, as the purpose of this experiment was to detect differences between different ES cell lines under normal tissue culture conditions. All cell lines were at passage 23 when sampled.
Growth protocol
ES cells were maintained in their undifferentiated state in the medium MEM-Alpha (GibcoTM, Invitrogen Ltd, Paisley, Renfrewshire, UK) supplemented with 10-4M -Mercaptoethanol (Merck KGaA, 64293 Darmstadt, Germany), 2mM glutamine and 10-3 U/ml murine LIF (ESGROTM, Invitrogen, Ltd, Paisley, Renfrewshire, UK), 10% FBS and 10% NBS (selected batches, PAA Laboratories GmbH, Linz, A-4020 Austria). The cells were maintained at 37oC in a humidified atmosphere with 5% CO2 on 0.1% gelatin (Stem Cell Technologies) coated tissue culture grade plastic ware (NUNCTM, Fisher Scientific, Loughborough, Leics, UK).
Extracted molecule
cytoplasmic RNA
Extraction protocol
ES cells were washed twice with DPBS-A and treated with trypsin-EDTA (Invitrogen Ltd., Paisley, UK). The cells were counted in a haemocytometer and appropriate cell numbers were pelleted by centrifugation at 200g. The QIAgen RNeasyTM Midi Kit (Qiagen Ltd., Crawley, Sussex, UK) was used according to the manufacturer’s protocol for RNA extraction. These RNA samples were quantified and checked for degradation via OD 260/280 spectrophotometry (Camspec, Cambridge, UK) and gel electrophoresis using dissociating conditions (NorthernMax™ buffers; Ambion, Huntingdon, UK), used according to the manufacturer’s protocol.
Label
Cy5
Label protocol
Different RNA samples were used for labelling array repetitions. 10ug of total RNA was labelled with either Cy3 or Cy5 dyes using the CyScribe labelling system (GE Healthcare, Chalfont St.Giles, Bucks, UK), according to the manufacturer’s protocol. 1ul of labelled cDNA was combined with 2ul 50% glycerol, run on a ‘John gel’ (a microscope slide sized, 1.5% agarose gel) and scanned using a GeneTac LS IV scanner (Genomic Solutions, Huntingdon, Cambs. UK) in order to assess successful incorporation of label. Control and experimental samples were then combined and prepared for hybridization.
Channel 2
Source name
Pooled control, pool of HM1, IMT11 and SHBl6.3 undifferentiated mouse ES cell lines, p23
Pooled control consisting of equal amounts of RNA from 3 undifferentiated mouse ES cell lines, HM1, IMT11 and SHBl6.3, all at passage 23
Biomaterial provider
Cardiff University, Mrs Susan Hunter
Treatment protocol
See growth protocol. These ES cell lines were maintained normally, as the purpose of this experiment was to detect differences between different ES cell lines under normal tissue culture conditions. All cell lines were at passage 23 when sampled.
Growth protocol
ES cells were maintained in their undifferentiated state in the medium MEM-Alpha (GibcoTM, Invitrogen Ltd, Paisley, Renfrewshire, UK) supplemented with 10-4M -Mercaptoethanol (Merck KGaA, 64293 Darmstadt, Germany), 2mM glutamine and 10-3 U/ml murine LIF (ESGROTM, Invitrogen, Ltd, Paisley, Renfrewshire, UK), 10% FBS and 10% NBS (selected batches, PAA Laboratories GmbH, Linz, A-4020 Austria). The cells were maintained at 37oC in a humidified atmosphere with 5% CO2 on 0.1% gelatin (Stem Cell Technologies) coated tissue culture grade plastic ware (NUNCTM, Fisher Scientific, Loughborough, Leics, UK).
Extracted molecule
cytoplasmic RNA
Extraction protocol
ES cells were washed twice with DPBS-A and treated with trypsin-EDTA (Invitrogen Ltd., Paisley, UK). The cells were counted in a haemocytometer and appropriate cell numbers were pelleted by centrifugation at 200g. The QIAgen RNeasyTM Midi Kit (Qiagen Ltd., Crawley, Sussex, UK) was used according to the manufacturer’s protocol for RNA extraction. These RNA samples were quantified and checked for degradation via OD 260/280 spectrophotometry (Camspec, Cambridge, UK) and gel electrophoresis using dissociating conditions (NorthernMax™ buffers; Ambion, Huntingdon, UK), used according to the manufacturer’s protocol.
Label
Cy3
Label protocol
Different RNA samples were used for labelling array repetitions. 10ug of total RNA was labelled with either Cy3 or Cy5 dyes using the CyScribe labelling system (GE Healthcare, Chalfont St.Giles, Bucks, UK), according to the manufacturer’s protocol. 1ul of labelled cDNA was combined with 2ul 50% glycerol, run on a ‘John gel’ (a microscope slide sized, 1.5% agarose gel) and scanned using a GeneTac LS IV scanner (Genomic Solutions, Huntingdon, Cambs. UK) in order to assess successful incorporation of label. Control and experimental samples were then combined and prepared for hybridization.
Hybridization protocol
Array slides were incubated in prehybe for 1 hour at 42oC (50% formamide, 5XSSC, 0.1% SDS, 1% BSA). Targets were dried down via vacuum centrifugation then resuspended in 50l hybe solution (49.9% de-ionised formamide, 49.9% 20xSSC, 0.2% SDS) with added 1l Cot1 DNA and 1l poly A oligo as blocking agents, heated to 95oC for 5 minutes and then added to the face of one slide. The printed face of the second slide of the pair was then placed face to face with the first, using the same probe. Slide pairs were then placed on a level plastic cover above some 1xSSC moistened tissue in a slide box. The slide box was sealed with Nescofilm (Karlan Research Products Corporation, Santa Rosa, CA, USA), placed floating in a waterbath and hybridized for 24-48 hours at 42oC. Following hybridization, slides were washed once in Wash solution 1 (1X SSC, 2% SDS, filtered autoclaved ddH2O) for 20 minutes, then twice in Wash solution 2 (0.1X SSC, 0.2% SDS, filtered autoclaved distilled deionised H2O (ddH2O) for 20 minutes each. Slides were dipped in nuclease free filtered water, then spray dried, finally, the backs of the slides were cleaned with filtered autoclaved ddH2O, then wiped with 100% EtOH, then wiped dry and scanned.
Scan protocol
Scans were carried out at 12.5 micrometers, using the averaging setting (GeneTac LSIV scanner, (Genomic Solutions, Cambridgeshire, UK)). It is possible to carry out quick draft scans using this scanner, gain and black settings, which affect image intensity and background were varied slightly in order to optimize the signal/noise ratio for each channel and each slide before proper scans were initiated.
Description
Arrays were repeated 12 times with fluor switching, in order to counteract any issues of dye bias that may have arisen from direct labelling. 6 biological repeats were carried out (6 ES cell RNA samples for each ES cell line) Scanned images were stored and filtered, then analysed using the program ImaGene™ 5.5 (BioDiscovery). Analysis and other methods were carried out similarly to another published experiment, please see below and Mansergh et al. 2004 PMID: 15303089 for more details.
Data processing
Output files from ImaGene were saved in MS Excel spreadsheet format. MS Excel was used for all further data manipulation. We normalised each channel via total array methods, via calculation of the mean intensity value. Normalised intensities were then analysed using two different methods. Firstly, we transferred normalised intensity values for each experimental repetition into a separate Excel file, ensuring a standard order of samples. These data were then formatted for Significance Analysis of Microarrays (SAM; http://www-stat.stanford.edu/~tibs/SAM ). SAM analysis was carried out and genes that showed a fold change of 2 or above and that appeared in a SAM analysis as statistically significant above a delta value of 0.5 (which denotes an error rate of 5%) were selected for further appraisal. We supplemented SAM analysis with a second analysis method as follows: We subtracted the background median from the signal median, removing values of less than zero using the following equation :IF(k2>0,k2,0.0001), where k2 is the row and column number of the background subtracted data column. This equation replaced any negative values with 0.0001. The data column was then normalised by division by the mean, resulting in values >1 being above average intensity and vice versa. The mean background + 2 standard deviations of the background were calculated from the median background column, and divided by the normalisation factor calculated above. Fold changes were calculated for control and experimental data, furthermore, datapoints that were below background + 2sd were rejected. Meaningful data (above background + 2SD values, fold change of > 2) were retained, combined with similar data from other replicates and sorted via accession number. Genes that appeared above background + 2SD values with a fold change of > 2, in at least 9 out of 12 replicates were retained for further analysis. Finally, accession numbers obtained from this method and from SAM were compared. Genes appearing as differentially regulated using BOTH methods were deemed significant by us. In other words, significant ESTs are changed in expression by at least two fold, are above background + 2SD in at least one channel and have a delta value of 0.5 using SAM. These ESTs were subjected to bioinformatic analysis and were ALL assessed via RT PCR in order to confirm array results.
