Investigation Title Transcription profiling of mouse Icmt+/+ and Icmt-/- embryonic fibroblasts cells
Comment[Submitted Name] DJVaux_MEF_Icmt
Experimental Design cell_type_comparison_design transcription profiling by array
Experimental Design Term Source REF mo EFO
Comment[ArrayExpressReleaseDate] 2007-01-08
Comment[AEMIAMESCORE] 4
Comment[ArrayExpressAccession] E-MEXP-540
Comment[MAGETAB TimeStamp_Version] 2011-01-23 09:50:13 Last Changed Rev: 14857
Experimental Factor Name genotype
Experimental Factor Type genotype
Experimental Factor Term Source REF
Person Last Name Saunders
Person First Name Nigel
Person Mid Initials J
Person Email nigel.saunders@path.ox.ac.uk
Person Phone 01865 275521
Person Fax 01865 275515
Person Address South Parks Road
Person Affiliation Sir William Dunn School of Pathology
Person Roles submitter
Person Roles Term Source REF
Quality Control Type
Quality Control Term Source REF
Replicate Type
Replicate Term Source REF
Normalization Type
Normalization Term Source REF
Date of Experiment
Public Release Date 2007-01-08
PubMed ID 17312019
Publication DOI 17312019
Publication Author List Ashraf Malhas, Chiu Fan Lee, Rebecca Sanders, Nigel J. Saunders, David J. Vaux
Publication Title Defects in lamin B1 expression or processing affect interphase chromosome position and gene expression
Publication Status journal_article
Publication Status Term Source REF
Experiment Description Comparison of gene expression profiles of Icmt+/+ and Icmt-/- cells
Protocol Name P-MEXP-9540 P-MEXP-9537 P-MEXP-16921 P-MEXP-16920 P-MEXP-16145 P-MEXP-9545
Protocol Type grow nucleic_acid_extraction labeling hybridization feature_extraction bioassay_data_transformation
Protocol Description Mouse embryonic fibroblasts (MEFs); Rce1+/+, Rce1-/-, Lmnb1+/+, Lmnb1-/-, Icmt+/+ and Icmt-/- were cultured in DMEM supplemented with 10% FCS, L-glutamine and non-essential amino acids at 37 degrees C in a humidified atmosphere. Cells were grown in 25cm2 flasks, seeded at 50% confluency and RNA was extracted when cells reached ~90% confluency. RNA Extraction: TriZol/RNEasy Method:
TriZol Extraction and RNEasy Cleanup:
1. Add TriZol to the cell monolayer at 1 ml per 1 square cm of tissue culture surface.
2. Incubate at room temperature for 5 minutes.
3. Add 200µl chloroform and shake for 15 seconds.
4. Incubate at room temperature for 2-3 minutes.
5. Centrifuge at 12 000 xg at 4C for 15 minutes.
6. Transfer the colourless upper aqueous phase (containing the RNA) to a new RNAse free tube.
7. Add 500µl of isopropanol and incubate at room temperature for 10 minutes. The addition of 1µl of GlycoBlue (Ambion) allows better visualisation of the precipitated RNA pellet if small quantities of RNA are expected.
8. Centrifuge at 12 000 xg at 4oC for 10 minutes.
9. Remove as much of the supernatant from the pellet as possible and resuspend the pellet in 100µl H2O
RNEasy Cleanup:
1. Add 350µl RLT buffer (note: 10µl ß-ME needs to be added per 1ml buffer RLT prior to use) and mix thoroughly
2. Add 250µl absolute ethanol to sample and mix well by pipetting up and down.
3. Apply sample to an RNEasy mini spin-column sitting in a 2ml collection tube. Centrifuge for 15 seconds at 8000 xg.
4. Pass the sample eluate over the column again.
5. Transfer the spin-column to a new 2ml collection tube and add 500µl Buffer RPE and centrifuge for 15 seconds at 8000 xg to wash.
6. Discard the flow through and add a further 500µl Buffer RPE onto the spin-column and centrifuge for 2 minutes at 8000 xg to dry the membrane.
7. Place the spin column in a new 1.5ml microfuge tube and pipette 70µl RNAse free H2O directly onto the membrane. Allow to stand for 1 minute. Centrifuge for 1 minute at 8000 x g to elute.
8. Sub aliquot the sample based upon the concentration so that replicate samples are generated so each is only frozen once.
9. Add 1µl of Superase.In RNAse inhibitor to each sample and place it on dry ice or transfer to -80 IMMEDIATELY.
BPFG direct amino-allyl dUTP microarray labelling protocol Day 1 Spin down a new tube each of monoreactive Cy3 and Cy5. For 6 slides (2 sets of 3, the third of each set is a dye swap) add 80 l of fresh DMSO from a vial to the Cy3 and Cy5 tubes and resuspend the dye. Add 1 molecular sieve bead to each tube of Cy dye. The tubes can then be refrozen in the foil pouch until ready to use. Defrost Random Primers, HySpot amino alyl dNTP, 5x First Strand Buffer, 0.1 M DTT, and RNA. Defrost a 5 ml aliquot of BSA. Check the pH of the NaCO3 (pH 9). Set PCR machine to 25°C for 2 min., 25°C for 10 min., 42°C for 90 min., 65°C for 15 min., hot start at 25°C. Set the incubator at 42°C. Set the hybridization oven to 65°C. Make a master mix: MIX 2 rxns 4rxns 6 rxns Random primers 2 l 4 l 8 l 12 l HySpot dNTP 1.5 l 3 l 6 l 9 l 5x First Strand Bfr 6 l 12 l 24 l 36 l 0.1 M DTT 3 l 6 l 12 l 18 l 25 l 50 l 75 l Set up the Reverse Transcriptase reactions in 0.2 ml tubes using 5-15 g of RNA: Control Test RNA + RNase free water 16.5 l 16.5 l MIX 12.5 l 12.5 l 29 l 29 l Mix each tube with pipette tip. Put tubes in PCR machine and run at 25°C for 2 minutes. When PCR machine steps to 25°C for 10 minutes, add 1 l of SuperScript III Reverse Transcriptase during that time, mixing with the pipette tip. PCR machine will then step to 42°C for 90 minutes. At the end of 90 minutes at 42°C pause the PCR machine program and add 10 l of 1 M NaOH. Resume PCR machine program for the 65°C step for 15 minutes. Set up and label the DNA Clean and Concentrator columns and tubes. Remove the tubes from the PCR machine and set it to hold at 25°C. Take the tubes of Cy dye out of the freezer, spin down, and keep them in the dark. Add 10 l of 1 M HCl to the tubes. Add 100 l DNA Binding Buffer from the DNA Clean & Concentrator Kit to the tubes. Transfer the reactions to the DNA Clean & Concentrator columns, labelled and in a collection tube. Spin 10 seconds. Pour out contents of collection tube. Add 200 l of Wash 1 to the column (this is not the wash included in the kit). Spin 10 seconds. Pour out contents of collection tube. Add 200 l of Wash 1 to the column. Spin 30 seconds. Put the column in a fresh, labelled recovery tube. Elute with 6 l of MilliQ, spinning briefly. Elute with a second round of 6 l of MilliQ and a brief spin. Transfer the ~12 l to a 0.2 ml tube. Add 1.2 l of 1 M NaCO3 pH 9. OPTION A: For Cy dye labelling: Add 13.2 l of Cy5 to the Control reaction for the first 2 slides, or to the Test for the third slide dye swap. Add 13.2 l of Cy3 to the Test reaction for the first 2 slides, or to the Control for the third slide dye swap. Remember to keep the Cy dye tubes and these reaction tubes in the dark as much as possible the dye is sensitive to light. Put the tubes at 25°C for 1 hour in the PCR machine to couple the dye to the incorporated amino alyl dUTPs. During this time pre-hybridize your slide (if needed) and prescan your slide (cleaning it if needed). Set up and label the DNA Clean and Concentrator columns and tubes. At the end of the hour, transfer the reactions to 1.5 ml tubes, keeping them out of the light. For both reactions: Set the PCR machine to hold at 42°C. Add 105.6 l 100 mM NaOAc pH 5.2. Add 264 l DNA Binding Buffer to the reactions. Transfer the reactions to the DNA Clean & Concentrator columns, labelled and in a collection tube. Spin 10 seconds. Pour out contents of collection tube. Do not be alarmed that this is very brightly coloured! Add 200 l of Wash 2 to the column (this is the wash supplied in the kit). Spin 10 seconds. Pour out contents of collection tube. Add 200 l of Wash 2 to the column. Spin 30 seconds. Put the column in a fresh, labelled recovery tube. Elute with 6 l of MilliQ, spinning briefly. Elute with a second round of 6 l of MilliQ and a brief spin. The elutions should retain some of the colour seen in the reactions. Mix the ~12 l Cy5 labelled reaction with the ~12 l Cy3 labelled reaction, transferring to a 0.2 ml tube. Add 24 l Gen HYB hybridization buffer to the Cy mixture. Heat the hybridization mixture to 42°C in the PCR machine. Put three staining troughs, Gen HYB Wash A, Gen HYB Wash B, and Gen HYB Wash C at 42°C in the incubator. Remove the pre-warmed slide in its chamber from the incubator and place on a piece of foil large enough to wrap the chamber. Pipette the 48 l of 42°C hybridization mixture under the Lifter Slip, or onto the slide and add a coverslip. Adjust this volume according to required volumes for larger LifterSlips / arrayed areas using 1x GenHyb solution, prepared from 2x stock GenHyb and MilliQ water. Add the appropriate amount of MilliQ for the hybridization chamber (10 l each of 2 wells in the Corning chambers) and seal the chamber. Wrap the chamber in foil to exclude light and place in the 42°C incubator overnight. Or for Alexa labelling: aa-cDNA is labelled with the Alexa dye- ARES kit (Molecular Probes) according to the manufacturers instructions. Briefly: For each fluorophore (typically for Alexa fluor 555 and Alexa fluor 647, for a Cy-3 and Cy5 equivalent stimulation / emission lablling), processed in parallel: Prepare labelling buffer: Add 1 mL RNAse free water (Component E) to the vial of sodium bicarbonate and vortex (This can be stored at -20oC for up to 6 months) Warm DMSO (Component C), labelling buffer, and RNAser free water to RT Add 3 µl of Labelling Buffer to the 5 µl of aa-cDNA solution Prepare the reactive dye by dissolving 1 vial of reactive dye (Component B) in 5µl of DMSO (Component C) and vortex for 10 seconds (sufficient for 2 labelling reactions done on the same day) Add 2.5µl of dissolved reactive dye to the aa-cDNA/Labelling Buffer mix, vortex briefly, and incubate in the dark at room temperature for 1 hour Combine the labelled cDNAs (Alexa-555 and 647) which will generate a final volume of 21 µl. Set up and label the DNA Clean and Concentrator columns and tubes. Set the PCR machine to hold at 42°C. Add 200 l DNA Binding Buffer to the reactions. Transfer the reactions to the DNA Clean & Concentrator columns, labelled and in a collection tube. Spin 10 seconds. Pour out contents of collection tube. Do not be alarmed that this is very brightly coloured! Add 200 l of Wash 2 to the column (this is the wash supplied in the kit). Spin 10 seconds. Pour out contents of collection tube. Add 200 l of Wash 2 to the column. Spin 30 seconds. Put the column in a fresh, labelled recovery tube. Elute with 6 l of MilliQ, spinning briefly. Elute with a second round of 6 l of MilliQ and a brief spin. The elutions should retain some of the colour seen in the reactions. Mix the ~12 l Cy5 labelled reaction with the ~12 l Cy3 labelled reaction, transferring to a 0.2 ml tube. Add 24 l Gen HYB hybridization buffer to the Cy mixture. BPFG direct amino-allyl dUTP microarray labelling protocol with GenHyb-based hybridization (2003) Day 1 Spin down a new tube each of monoreactive Cy3 and Cy5. For 6 slides (2 sets of 3, the third of each set is a dye swap) add 80 l of fresh DMSO from a vial to the Cy3 and Cy5 tubes and resuspend the dye. Add 1 molecular sieve bead to each tube of Cy dye. The tubes can then be refrozen in the foil pouch until ready to use. Defrost Random Primers, HySpot amino alyl dNTP, 5x First Strand Buffer, 0.1 M DTT, and RNA. Defrost a 5 ml aliquot of BSA. Check the pH of the NaCO3 (pH 9). Set PCR machine to 25°C for 2 min., 25°C for 10 min., 42°C for 90 min., 65°C for 15 min., hot start at 25°C. Set the incubator at 42°C. Set the hybridization oven to 65°C. Make a master mix: MIX 2 rxns 4rxns 6 rxns Random primers 2 l 4 l 8 l 12 l HySpot dNTP 1.5 l 3 l 6 l 9 l 5x First Strand Bfr 6 l 12 l 24 l 36 l 0.1 M DTT 3 l 6 l 12 l 18 l 25 l 50 l 75 l Set up the Reverse Transcriptase reactions in 0.2 ml tubes using 5-15 g of RNA: Control Test RNA + RNase free water 16.5 l 16.5 l MIX 12.5 l 12.5 l 29 l 29 l Mix each tube with pipette tip. Put tubes in PCR machine and run at 25°C for 2 minutes. When PCR machine steps to 25°C for 10 minutes, add 1 l of SuperScript III Reverse Transcriptase during that time, mixing with the pipette tip. PCR machine will then step to 42°C for 90 minutes. At the end of 90 minutes at 42°C pause the PCR machine program and add 10 l of 1 M NaOH. Resume PCR machine program for the 65°C step for 15 minutes. Set up and label the DNA Clean and Concentrator columns and tubes. Remove the tubes from the PCR machine and set it to hold at 25°C. Take the tubes of Cy dye out of the freezer, spin down, and keep them in the dark. Add 10 l of 1 M HCl to the tubes. Add 100 l DNA Binding Buffer from the DNA Clean & Concentrator Kit to the tubes. Transfer the reactions to the DNA Clean & Concentrator columns, labelled and in a collection tube. Spin 10 seconds. Pour out contents of collection tube. Add 200 l of Wash 1 to the column (this is not the wash included in the kit). Spin 10 seconds. Pour out contents of collection tube. Add 200 l of Wash 1 to the column. Spin 30 seconds. Put the column in a fresh, labelled recovery tube. Elute with 6 l of MilliQ, spinning briefly. Elute with a second round of 6 l of MilliQ and a brief spin. Transfer the ~12 l to a 0.2 ml tube. Add 1.2 l of 1 M NaCO3 pH 9. OPTION A: For Cy dye labelling: Add 13.2 l of Cy5 to the Control reaction for the first 2 slides, or to the Test for the third slide dye swap. Add 13.2 l of Cy3 to the Test reaction for the first 2 slides, or to the Control for the third slide dye swap. Remember to keep the Cy dye tubes and these reaction tubes in the dark as much as possible the dye is sensitive to light. Put the tubes at 25°C for 1 hour in the PCR machine to couple the dye to the incorporated amino alyl dUTPs. During this time pre-hybridize your slide (if needed) and prescan your slide (cleaning it if needed). Set up and label the DNA Clean and Concentrator columns and tubes. At the end of the hour, transfer the reactions to 1.5 ml tubes, keeping them out of the light. For both reactions: Set the PCR machine to hold at 42°C. Add 105.6 l 100 mM NaOAc pH 5.2. Add 264 l DNA Binding Buffer to the reactions. Transfer the reactions to the DNA Clean & Concentrator columns, labelled and in a collection tube. Spin 10 seconds. Pour out contents of collection tube. Do not be alarmed that this is very brightly coloured! Add 200 l of Wash 2 to the column (this is the wash supplied in the kit). Spin 10 seconds. Pour out contents of collection tube. Add 200 l of Wash 2 to the column. Spin 30 seconds. Put the column in a fresh, labelled recovery tube. Elute with 6 l of MilliQ, spinning briefly. Elute with a second round of 6 l of MilliQ and a brief spin. The elutions should retain some of the colour seen in the reactions. Mix the ~12 l Cy5 labelled reaction with the ~12 l Cy3 labelled reaction, transferring to a 0.2 ml tube. Add 24 l Gen HYB hybridization buffer to the Cy mixture. Heat the hybridization mixture to 42°C in the PCR machine. Put three staining troughs, Gen HYB Wash A, Gen HYB Wash B, and Gen HYB Wash C at 42°C in the incubator. Remove the pre-warmed slide in its chamber from the incubator and place on a piece of foil large enough to wrap the chamber. Pipette the 48 l of 42°C hybridization mixture under the Lifter Slip, or onto the slide and add a coverslip. Adjust this volume according to required volumes for larger LifterSlips / arrayed areas using 1x GenHyb solution, prepared from 2x stock GenHyb and MilliQ water. Add the appropriate amount of MilliQ for the hybridization chamber (10 l each of 2 wells in the Corning chambers) and seal the chamber. Wrap the chamber in foil to exclude light and place in the 42°C incubator overnight. Or for Alexa labelling: aa-cDNA is labelled with the Alexa dye- ARES kit (Molecular Probes) according to the manufacturers instructions. Briefly: For each fluorophore (typically for Alexa fluor 555 and Alexa fluor 647, for a Cy-3 and Cy5 equivalent stimulation / emission lablling), processed in parallel: Prepare labelling buffer: Add 1 mL RNAse free water (Component E) to the vial of sodium bicarbonate and vortex (This can be stored at -20oC for up to 6 months) Warm DMSO (Component C), labelling buffer, and RNAser free water to RT Add 3 µl of Labelling Buffer to the 5 µl of aa-cDNA solution Prepare the reactive dye by dissolving 1 vial of reactive dye (Component B) in 5µl of DMSO (Component C) and vortex for 10 seconds (sufficient for 2 labelling reactions done on the same day) Add 2.5µl of dissolved reactive dye to the aa-cDNA/Labelling Buffer mix, vortex briefly, and incubate in the dark at room temperature for 1 hour Combine the labelled cDNAs (Alexa-555 and 647) which will generate a final volume of 21 µl. Set up and label the DNA Clean and Concentrator columns and tubes. Set the PCR machine to hold at 42°C. Add 200 l DNA Binding Buffer to the reactions. Transfer the reactions to the DNA Clean & Concentrator columns, labelled and in a collection tube. Spin 10 seconds. Pour out contents of collection tube. Do not be alarmed that this is very brightly coloured! Add 200 l of Wash 2 to the column (this is the wash supplied in the kit). Spin 10 seconds. Pour out contents of collection tube. Add 200 l of Wash 2 to the column. Spin 30 seconds. Put the column in a fresh, labelled recovery tube. Elute with 6 l of MilliQ, spinning briefly. Elute with a second round of 6 l of MilliQ and a brief spin. The elutions should retain some of the colour seen in the reactions. Mix the ~12 l Cy5 labelled reaction with the ~12 l Cy3 labelled reaction, transferring to a 0.2 ml tube. Add 24 l Gen HYB hybridization buffer to the Cy mixture. Heat the hybridization mixture to 42°C in the PCR machine. Put three staining troughs, Gen HYB Wash A, Gen HYB Wash B, and Gen HYB Wash C at 42°C in the incubator. Remove the pre-warmed slide in its chamber from the incubator and place on a piece of foil large enough to wrap the chamber. Pipette the 48 l of 42°C hybridization mixture under the Lifter Slip, or onto the slide and add a coverslip. Adjust this volume according to required volumes for larger LifterSlips / arrayed areas using 1x GenHyb solution, prepared from 2x stock GenHyb and MilliQ water. Add the appropriate amount of MilliQ for the hybridization chamber (10 l each of 2 wells in the Corning chambers) and seal the chamber. Wrap the chamber in foil to exclude light and place in the 42°C incubator overnight. PREHYBRIDIZATION OF SLIDES (if needed) In a Coplin jar add: 5 ml BSA (100 mg/ml) 8.75 ml 20x SSC 250 l 20% SDS up to 50 ml with MilliQ (does not need to be exact, mark ~50 ml on your Coplin jar and go by that) Put the Coplin jar and prehybridization solution in the 65°C oven until it is up to temperature. Put the slide in the 65°C prehybridization solution in the Coplin jar in the oven for 20 minutes. Rinse the slide in a trough of MilliQ for 1 minute. Rinse the slide in a trough of isopropanol for 1 minute. (can be re-used about 5 times) Dry the slide, scan it, clean it more in the isopropanol if needed, and put it in a hybridization chamber with a Lifter Slip at 42°C in the incubator. Day 2 Take the Wash A trough and GenHyb Wash A solution out of the incubator and place the trough on a piece of foil large enough to wrap the trough. Fill the trough with GenHyb Wash A. Remove the slides from their hybridization chambers and float the LifterSlips off of them in the GenHyb Wash A. Load the slides into the staining rack, secure them with a rubber band, and place them in the GenHyb Wash A trough. Wrap with foil and place in the 42°C incubator for 5 minutes. Take the Wash B trough and GenHyb Wash B solution out of the incubator and fill the trough with GenHyb Wash B. Unwrap the GenHyb Wash A trough and place the GenHyb Wash B trough on the foil. Transfer the staining rack with the slides to the GenHyb Wash B trough. Wrap with foil and place in the 42°C incubator for 5 minutes. Take the Wash C trough and GenHyb Wash B solution out of the incubator and fill the trough with GenHyb Wash C. Unwrap the GenHyb Wash B trough and transfer the staining rack with the slides to the GenHyb Wash C trough. Wash the slides for 30 seconds. Dip the slide briefly in MilliQ, dry it quickly, and scan it. Wash 1 GenHyb Wash A 50 ml 20x SSC 1x SSC 10 ml 20% SDS 0.2% SDS to 1 litre with MilliQ GenHyb Wash B 10 ml 20x SSC 0.2x SSC 10 ml 20% SDS 0.2% SDS to 1 litre with MilliQ GenHyb Wash C 5 ml 20x SSC 0.1x SSC to 1 litre with MilliQ Standard Solutions needed: 1 M NaOH 1 M HCl 1 M NaCO3 pH 9 100 mM NaOAc pH 5.2 100 mg/ml BSA (freeze in 5 ml aliquots) 20x SSC 20% SDS Special materials to buy: Cy3 mono-reactive dye pack Amersham PA23001 Cy5 mono-reactive dye pack Amersham PA25001 Or ARES Alexa flour 555 DNA labelling kit Molecular Probes A-21676 ARES Alexa flour 647 DNA labelling kit Molecular Probes A-21677 di-methylsulfoxide (DMSO) 5 x 5ml vials Sigma D2650 It is very important that a fresh vial is used to ensure that no water has entered solution. Molecular sieve grade 3A, 1.6-2.5 mm, 500 g GeeJay Chemicals Ltd. One bead is added to opened vials of Cy dyes to prolong their shelf life after opening. Random Primers 3 g/l Invitrogen 48190-011 SuperScript III Reverse Transcriptase Invitrogen 18080044 includes 5x First Strand Buffer and 0.1 M DTT HySpot amino alyl dUTP in dNTP mix 100 l Genetix K2702 DNA Clean & Concentrator kit Genetix D4003 Gen HYB hybridization buffer Genetix K2105 Equipment/materials needed: Programmable PCR machine We use a BIORAD iCycler. 65°C oven 42°C incubator Micro-centrifuge Microarray slides This protocol has been optimised for use with cDNA slides, but should also work well for oligo slides, although we have not tested it. Lifter Slips or other slide coverslips LifterSlips are difficult to get in the UK. VWR is supposed to supply them. We had to order them directly from the US from Eire Scientific. Slide hybridization chambers We use Corning hybridization chambers, but many others are good as well. Staining troughs Coplin jars Slide Staining racks Dark slide boxes for storage/transport Microarray slide scanner and analysis software We use an Axon 4000B with GenePix software for scanning and data acquisition and GeneSpring for analysis of the data. Foil 1.5 tubes 0.2 ml tubes (or the size to fit your PCR machine) Scanned using 10 micron setting, 100% laser power, and varied the PMT setting to just below saturation. This is an efficient C++ implementation by Björn Samuelsson of the LOWESS algorithm. It provides intensity-based normalization.
The window size is the only smoothness parameter available in this implementation. The higher it is, the smoother the function will be. Tweaking the other two parameters allows you to gain performance at the price of a small precision loss. The default values are usually good. Note that LOWESS can't handle spots with negative intensities, so these will be filtered out.
Protocol Parameters media;start time;min temperature; Amplification;Extracted product; Amplification;Label used; Chamber type;temperature;Quantity of label target used;
Protocol Hardware ScanArray Express HT [PerkinElmer]
Protocol Software ScanArray Express [PerkinElmer]
Protocol Contact
Protocol Term Source REF
SDRF File E-MEXP-540.sdrf.txt
Term Source Name ncbitax ArrayExpress mo EFO
Term Source File http://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html http://www.ebi.ac.uk/arrayexpress http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/efo/
Term Source Version