9 protocols
AccessionNameType
P-UMCU-42
feature_extraction
Scanning was performed in a temperature- and humidity-controlled laboratory (20 degrees Celsius, 38% humidity), filtered to remove ozone which was monitored to ensure that this was lower than 5 ppb. Slides were scanned using a G2565BA scanner that has a 48 slide carousel (Agilent, California, USA) at 100% laser power and 30% PMT. After scanning, the intensities for the Cy5 (Red) and Cy3 (Green) channels were automatically extracted using the batch-processing module in ImaGene version 8.0.1 (Biodiscovery, California, USA). For spot finding a local flexibility of 2.0 pixels was used. For segmentation the following settings were used: background buffer: 3.0; background width: 3.0; signal percentages: 3% (low), 97% (high); background percentages: 3% (low), 97% (high). Measurements exported: mean, median, total, standard deviation and area of the foreground and background signals. Batch editor configuration files are available upon request.
P-UMCU-38
labeling
All robotic RNA amplification and labelling procedures were performed in 96 well plates (4titude, Bioke) on a customized Sciclone ALH 3000 Workstation (Caliper LifeSciences) that included a PCR PTC-200 (Bio-Rad Laboratories), SpectraMax 190 spectrophotometer (Molecular Devices), and a magnetic bead-locator (Beckman). For a movie of this automated setup see: http://microarrays.holstegelab.nl/modx/index.php?id=66
RNA amplification
For RNA amplification, total RNA samples were diluted to 0.2 ug/ul and 5 ul is put in a 96-wells plate (Abgene).
All subsequent steps were performed by the following robot script:
1. Mix1 (5 ul) containing 100 ng T7 Mlu VN primer (5’ GGA GGC CGG AGA ATT GTA ATA CGA CTC ACT ATA GGG AGA CGC GTG TTT TTT TTT TTT TTT TTT TTT TTT VN 3’, whereby V= G, A or C; N= G, A, C or T) and external control RNAs, is added to the 5 ul total RNA and mixed in each well. Plate is incubated at 70 degrees C for 10 min and cooled to 48 degrees C.
2. Mix2 containing 4 ul 5x 1st strand buffer, 2 ul 0.1 M DTT (Invitrogen), 1 ul RNAse Inhibitor (Boehringer), 1 ul 20 mM dNTPs (GE Healthcare), 1 ul linear acrylamide, and 1 ul SuperScriptIII (Invitrogen) per sample is prewarmed to 48 degrees C. 10 ul per sample is added and mixed in each well.
3. Plate is incubated at 48 degrees C for 2 hours and cooled to room temperature.
4. 106 ul water and subsequently mix3 containing 15 ul second strand buffer, 3 ul 20 mM dNTPs (GE Healthcare), 1 ul T4 DNA ligase, 4 ul E.coli DNA polymerase I and 1 ul RNAseH (Promega) is added and mixed in each well.
5. Plate is incubated at 16 degrees C for 2 hours, at 65 degrees C for 10 minutes. Double-stranded cDNA product is purified and concentrated with RNAClean (Agencourt, GC biotech) according to manufacturers’ protocol, to an end volume of 25 ul.
6. 8 ul cDNA is put in a 96 well plate and mixed with 12 ul IVT mix containing 2 ul 10x rxn-buffer, 2 ul of each ATP, CTP, GTP, 0.6 ul UTP, 2 ul T7 enzyme mix (MegaScript kit, Ambion, Applied Biosystems), and 2.1 ul 50 mM 5-(3-aminoallyl)-UTP (Ambion, Applied Biosystems).
7. Plate is incubated at 37 degrees C for 4 hours. cRNA product is purified with RNAClean (Agencourt, Beckman) according to manufacturers’ protocol.
8. Concentration is measured (SpectraMax 190) and adjusted to 600 ng/ul. An aliquot of the resulting cRNA plates are sampled QC by Qiaxcel, the remainder is snapfrozen and stored at -80 degrees C.
Labelling
1. NHS-ester Cy3 or Cy5 dye (Amersham PA 23001 and 25001): entire tube is resuspended in 100 ul DMSO (Merck 8.02912.10).
2. 8 ul of each cRNA sample (0.6 ug/ul) is put in a 96-well plate (Abgene), the accompanying common reference sample is put in the next column (final step combines Cy3 and Cy5 labelled material).
3. 3 ul 0.5 M Sodium Bicarbonate buffer, pH 9 is added and mixed to all wells.
4. 3 ul Cy-dye solution is added and mixed to the appropriate wells, plate is incubated at 18 degrees C for 1 hour.
5. 4.5 ul 5 M hydroxylamine is added and mixed, incubated at 18 degrees C for 15 minutes. Labelled cRNA product is purified with RNA Clean (Agencourt, GC biotech) according to manufacturers' protocol.
6. RNA concentration and labelling incorporation are measured (SpectraMax 190).
7. 2.5 ug of each labelled sample and reference cRNA are pooled and subjected to fragmentation according to protocol (Ambion, Applied Biosystems), 15 minutes at 70 degrees C. Samples are stored at -20 degrees C until hybridisation.
P-UMCU-39
hybridization
2.5 ug of each labelled sample in a total volume of 60 ul is combined with 60 ul 2x-hybmix, containing 50% formamide, 10xSSC, 0.2% SDS, 200 ug/ml herring sperm DNA. The hybridisations were performed for 16 hours at 42 degrees Celsius in a HS4800Pro hybstation (Tecan, Männedorf, Switzerland) as detailed below. Hybridisation was performed in a temperature- and humidity-controlled laboratory (20 degrees Celsius, 38% humidity), filtered to remove ozone which was monitored to ensure that this was lower than 5 ppb.
1. 60 ul labelled sample is combined with 60 ul 2x-hybmix, containing 50% formamide, 10xSSC, 0.2% SDS, 200 ug/ml herring sperm DNA.
2. Hybridisations of are performed on an HS4800Pro Hybstation (Tecan, Männedorf, Switzerland).
3. Priming (30 seconds wash and 30 seconds soak) with 5xSSC, 0.1%SDS at 42 degrees Celsius.
4. Injection of pre-hyb mix: 5xSSC, 25% formamide, 0.1%SDS, 1%BSA, total volume 110 ul.
5. Pre-hybridisation: 45 minutes at 42 degrees Celsius, agitation frequency medium, other settings off.
6. Wash in milliQ (2 min wash, 1 min soak), at 42 degrees Celsius, 2x.
7. Wash in 5xSSC, 0.1%SDS (45 secs), at 42 degrees Celsius.
8. Injection of sample. Volume 110 ul.
9. Hybridisation: 16 hours at 42 degrees Celsius, agitation frequency medium, other settings off.
10. Wash (1 min wash, 1 min soak) in 1xSSC, 0.2%SDS at 23 degrees Celsius, 2x.
11. Wash (1 min wash, 1 min soak) in 0.1xSSC, 0.2%SDS at 23 degrees Celsius, 2x.
12. Wash (1 min wash, 1 min soak) in 0.1xSSC at 23 degrees Celsius, 4x.
13. Drying: blow with nitrogen for 3 min at 30 degrees Celsius.
P-UMCU-43
bioassay_data_transformation
Microarray data normalisation was performed on mean intensity values using print-tip LOESS as described and implemented in the marray R package version 1.20.0, using no background subtraction and a window span of 0.4. Probes flagged as absent, or with a (nearly) saturated signal (i.e., > 2^15) in either channel were not considered for the estimation of the LOESS curve. Gene-specific dye bias (GSDB) is not addressed by LOESS, and was corrected after the LOESS normalisation using the Gene- And Slide-Specific COrrection (GASSCO) algorithm implemented in the R package dyebias version 1.7.8. The correction consists of two components: the intrinsic dye bias of a specific probe and the degree of dye bias observed in a specific slide. The correction is based on the following formula:
Mij* = Mij + GSDBij = Mij + iGSDBi × Fj
Where Mij* is the measured (biased) log2 fold-change of gene i in hybridisation j, Mij is its unbiased version, GSDBij is the gene-specific dye bias component of Mij*.The latter is the product of the so-called intrinsic gene-specific dye bias of gene i (iGSDBi) and the slide bias of hybridisation j (Fj). The corrected log2 fold-change Mij is then calculated using:
Mij = Mij* - iGSDBi x Fj
The iGSDBs are estimated only once in a set of 200 wt vs. wt hybridisations (ArrayExpress accession E-TABM-773) and are used for all slides within this project. The slide bias F is estimated for each hybridisation separately, using two groups of probes, having the strongest red or green biases, defined as those with an iGSDB in the top and bottom 5th percentiles of iGSDBs, respectively. The slide bias is the mean of these red and green probe group's median (M / iGSDB)-ratio.
P-UMCU-40
image_acquisition
Scanning was performed in a temperature- and humidity-controlled laboratory (20 degrees Celsius, 38% humidity), filtered to remove ozone which was monitored to ensure that this was lower than 5 ppb. Slides were scanned using a G2565BA scanner that has a 48 slide carousel (Agilent, California, USA) at 100% laser power and 30% PMT. After scanning, the intensities for the Cy5 (Red) and Cy3 (Green) channels were automatically extracted using the batch-processing module in ImaGene version 8.0.1 (Biodiscovery, California, USA). For spot finding a local flexibility of 2.0 pixels was used. For segmentation the following settings were used: background buffer: 3.0; background width: 3.0; signal percentages: 3% (low), 97% (high); background percentages: 3% (low), 97% (high). Measurements exported: mean, median, total, standard deviation and area of the foreground and background signals. Batch editor configuration files are available upon request.
P-UMCU-37
nucleic_acid_extraction
use autoclaved MQ
1. Take Frozen cells (-80C) and resuspend in 500 ul Acid Phenol Chloroform (From Sigma, 5:1, pH 4.7). Immediately add the same volume TES-buffer (TES: 10 mM Tris pH 7.5, 10 mM EDTA, 0.5% SDS). Vortex very hard for 20 seconds (10 sec upright, 10 sec at an angle) to resuspend pellet.
2. Incubate in water bath for 10 minutes at 65C and vortex again.
3. Transfer to 1.5 ml Eppendorf-tubes. Put tubes in the thermomixer (65C, 1400RPM) for 50 minutes.
4. Spin for 20 minutes at 14000 rpm at 4C.
5. Take new Eppendorf-tube, fill with 500 ul Phenol Chloroform (5:1, pH 4.7). Add water-phase from point 5. Vortex hard for 20 seconds. Spin down for 10 minutes at 14000 rpm at 4C.
6. Take new Eppendorf-tube, fill with 500 ul Chloroform:Isoamyl-alcohol (25:1). Add water-phase from point 6. Vortex hard for 20 seconds. Spin down for 10 minutes at 14000 rpm at 4C.
7. Take new Eppendorf-tube, fill with 50 ul Sodium Acetate (NaAc 3M, pH 5.2). Add water-phase from point 7. Fill tube with Ethanol (100%, -20C). Flip tubes three times. Incubate at 20C for longer than 30 minutes.
8. Spin tubes down for 5 minutes at room temperature, 14000 rpm. Remove fluid with pipette. Be careful not to touch RNA-pellet.
9. Wash pellet with Ethanol 500 ul (80%, -20C). Spin down shortly.
10. Remove Ethanol, do not air dry but directly proceed to 11.
11. Dissolve RNA-pellet in 100ul sterile water (MQ, autoclaved). Pipet untill the pellet is released from the side of the tube. wait 5 minutes and pipet up and down 5 more times. Yield will be around 150ug. (Make sure all RNA is dissolved completely!)
12. measure concentration (dilute 100 times).
13. freeze in liquid N2 store at -80C.
P-UMCU-36
grow
S. cerevisiae BY4742 deletion mutants and the wild-type strains were cultured in SC Medium (Synthetic Complete; 4gr per 2l Drop out mix 13.42gr per 2l YNB(US biologicals) with 2% D-glucose)under agitation (230rpm), at 30C. The cells were collected at midlog.
P-UMCU-51
nucleic_acid_extraction
Total RNA was prepared by phenol extraction and cleaned up on a customized Caliper Sciclone ALH 3000 workstation that included a PCR machine PTC-200 (Bio-Rad Laboratories), SpectraMax 190 spectrophotometer (Molecular Devices) and a magnetic bead-locator (Beckman). For a movie of this automated setup see: http://microarrays.holstegelab.nl/modx/index.php?id=66
RNA extraction
Frozen cells (-80 degrees C) were resuspended in 500 ul acid phenol-chloroform (5:1, pH 4.7). An equal volume of TES-buffer (TES: 10 mM Tris pH 7.5, 10 mM EDTA, 0.5% SDS) was immediately added. Samples were vortexed very vigorously for 20 seconds and incubated in a water bath for 10 minutes at 65 degrees C and vortexed again. Samples were put in a shaking incubator (65 degrees C, 1400 rpm) for 50 minutes. Samples were centrifuged for 20 minutes at (20817 g at 4 degrees C. The aqueous phase was extracted and phenol extraction was repeated on this, followed by an otherwise identical chloroform:isoamyl-alcohol (25:1) extraction. RNA was precipitated with 50 ul sodium acetate (NaAc 3M, pH 5.2) and ethanol (96%, -20 degrees C), left at -20 degrees C for at least 30 minutes and centrifuged for 10 minutes at 20817 g at 4 degrees C. Pellet was washed with 80% ethanol (-20 degrees C) and dissolved in 90 ul sterile water (milliQ filtered), snapfrozen and stored at -80 degrees C.
RNA purification
1. Concentration of total RNA is measured by mixing 5 ul of the RNA sample with 45 ul water and determining the A260 from an absorbance spectrum on the SpectraMax.
2. 12.5 ul dnasei dilution (rnase-free dnase kit, qiagen, nr 79254, diluted 1:5 in rdd) is mixed with the remaining 87.5 ul rna-solution and incubated at 18 degrees C for 15 minutes.
3. RNA is purified and concentrated with RNAClean (Agencourt, Beckman) according to manufacturers’ protocol, to an end volume of 25 ul.
4. 5 ul cleaned RNA is mixed with 95 ul water and measured on the SpectraMAX, concentrations are normalised to 0.2 ug/ul in each well.
5. 5 ul of cleaned and normalised RNA is used to set up a start plate for RNA amplification.
6. 1 ul of cleaned and normalised RNA is used to check integrity by running on a capillary electrophoresis (QiaXcel, Qiagen) system.
7. All plates are snap frozen and stored at -80 degrees C until further use.
P-UMCU-48
bioassay_data_transformation
A software package for the analysis of gene expression microarray data, especially the use of linear models for analysing designed experiments and the assessment of differential expression. Author(s): Gordon Smyth. The limma R package version 2.12.0 is used. P-values are Benjamini-Hochberg FDR corrected.