Investigation Title Transcription profiling of Ky mouse mutant soleus muscles vs wild type soleus muscles
Comment[Submitted Name] MGU-Mouse-Ky mutant vs Wildtype
Experimental Design individual_genetic_characteristics_design transcription profiling by array
Experimental Design Term Source REF EFO
Comment[ArrayExpressReleaseDate] 2005-11-10
Comment[AEMIAMESCORE] 3
Comment[ArrayExpressAccession] E-MEXP-481
Comment[MAGETAB TimeStamp_Version] 2010-08-11 14:29:32 Last Changed Rev: 13058
Experimental Factor Name genotype
Experimental Factor Type genotype
Experimental Factor Term Source REF
Person Last Name Blanco Underhill
Person First Name Gonzalo Peter
Person Mid Initials
Person Email d.williams@har.mrc.ac.uk
Person Phone 01235 841135
Person Fax
Person Address
Person Affiliation Mammalian Genetics Unit, MRC Mammalian Genetics Unit, MRC
Person Roles biomaterial_provider submitter
Person Roles Term Source REF mo
Quality Control Type dye_swap_quality_control
Quality Control Term Source REF The MGED Ontology
Replicate Type
Replicate Term Source REF
Normalization Type
Normalization Term Source REF
Date of Experiment
Public Release Date 2005-11-10
PubMed ID
Publication DOI
Publication Author List Gonzalo Blanco; Peter Underhill
Publication Title Molecular phenotyping of the mouse ky mutant reveals UCP! upregulation at the neuromuscular junctions of dystrophic soleus muscle
Publication Status
Publication Status Term Source REF
Experiment Description A comparison of Ky mouse mutant soleus muscles versus wildtype soleus muscles. Hybridisation of 3 Mouse SGC 7.5k oligo slides, using 3 independent pools of 10 mice (5 male, 5 female)for both WT and mutant animals.
Protocol Name P-MEXP-14889 P-MEXP-14890 P-MEXP-7979 P-MEXP-7993 P-MEXP-7987 P-MEXP-7986 P-MEXP-7988 P-MEXP-7990
Protocol Type grow specified_biomaterial_action nucleic_acid_extraction pool labeling labeling hybridization image_acquisition
Protocol Description Ky is a spontaneous recessive mutation in the ky gene (Hum. Mol. Gen. 2001, Vol. 10, No. 1). It arose in the BDL albino genetic background. Homozygous ky/ky mice and normal control BDL were used for analysis of transcriptional changes in ky soleus muscle. Mice are maintained in the Mary Lyon Centre in ventilated cages in a temperature controlled (23 0C) virus-free facility on a 12 hour light/dark cycle and were fed a standard rodent chow diet (4RF21, Mucedola) and water ad libitum. At 45days old, sex matched mutant and control mice were killed by cervical dislocation. The soleus muscle was dissected immediately post-mortem from the hind limbs, snap-frozen in liquid nitrogen and kept at -80C until processed for RNA extraction. 1) Place the weighed (fresh, frozen, or RNAlater stabilized) tissue in a suitably sized vessel for the Rotor-stator homogenizer (e.g., in a 10-15 ml tube). Add the appropriate volume of Buffer RLT (see Table 1 below). Homogenize immediately using a conventional rotor-stator homogenizer for 45 s to 60 s until the sample is uniformly homogeneous
Note: Ensure that B-ME is added to Buffer RLT before use.
Table 1. Buffer RLT volumes for RNeasy Midi isolation of total RNA from animal
tissues
RNeasy column Amount of tissue (mg) Buffer RLT (ml)
Midi 20-75 2.0
Midi 75-130 2.0 (4.0)
Midi 130-250 4.0
2) Centrifuge the tissue lysate for 10 min at 3000-5000 x g. Carefully transfer the supernatant to a new 10-15 ml tube by pipetting. Use only this supernatant (lysate) in subsequent steps.
3) Add 1 volume (2.0 ml or 4.0 ml) of 70% ethanol to the homogenized lysate, and mix immediately by shaking vigorously. Ensure that any precipitates are resuspended. Do not centrifuge. Continue without delay with step 4.
4) Apply the sample to an RNeasy midi column placed in a 15 ml centrifuge tube and close the tube gently. Maximum loading volume is 4.0 ml. Centrifuge for 5 min at 3000-5000 x g. Discard the flow-through.
If the volume exceeds 4.0 ml, load aliquots successively onto the RNeasy column, and centrifuge as above. Discard the flow-through after each centrifugation step.
5) Pipet 2.0 Buffer RW1 into the RNeasy column. Centrifuge for 5 min at 3000-5000 x g to wash. Discard the flow-through.
6) Perform an on-column DNase digestion by adding 20 ul DNase I stock solution (dissolve the solid DNase I (1500 Kunitz units) in 550 ul of the RNase-free water. Mix gently by inverting the tube. Do not vortex.) to 140 ul Buffer RDD and mix by gently flicking the tube. Centrifuge the tube briefly to collect residual liquid from the sides of the tube. Pipet the DNase I incubation mix directly onto the RNeasy silica-gel membrane and place on the benchtop (20-30 C) for 15 min.
7) Pipet 2.0 ml Buffer RW1 into the RNeasy column, and place on the
benchtop for 5 min. Then centrifuge for 5 min at 3000-5000 x g. Discard the flowthrough.
8) Add 2.5 ml Buffer RPE to the RNeasy column. Close the centrifuge tube gently, and centrifuge for 2 min at 3000-5000 x g to wash the column. Discard the flow-through.
9) Add another 2.5 ml Buffer RPE to the RNeasy column. Close the centrifuge tube gently, and centrifuge for 5 min at 3000-5000 x g to dry the RNeasy silica-gel membrane.
10) To elute, transfer the RNeasy column to a new 15 ml collection tube. Pipet the appropriate volume of RNase-free water (see Table 2) directly onto the RNeasy silica-gel membrane. Close the tube gently. Let it stand for 1 min, and then centrifuge for 3 min at 3000-5000 x g.
Table 2. RNase-free water volumes for RNeasy Midi elution RNeasy column
Expected total RNA yield RNase-free water
Midi >150 ug 150 ul
Midi 150 ug - 1 mg 250 ul
11) Repeat the elution step (step 10) as described with a second volume of RNase-free water. To obtain a higher total RNA concentration, this second elution step may be performed using the first eluate (from step 11). The yield will be 15-30% less than the yield obtained using a second volume of RNase-free water, but the final concentration will be higher. Equal amounts of RNA were pooled from each sample Reverse Transcription
Total RNA x uL (50 ug total)
Oligo (dT) (T15) (0.5ug/uL) (Promega) 8 uL (4 ug total)
dH2O RNase-free (Sigma) to 24 uL
Heat to 70 0C x 5 min. and snap cool on Ice x 1min
Prepare the following:
[5x] RT buffer (BRL) 10 uL
0.1M DTT (BRL) 5 uL
25 mM dNTPs (including dCTP) 2 uL
RNase inhibitor (BRL) 1 uL
dH2O RNase-free (Sigma) 6 uL
Add the above mix to the RNA/Oligo (dT) solution & mix.
Add 2 uL of M-MLV II (Superscript II, BRL) & incubate at 42C. Add further 1 ul after each 60 minutes.
After 4 hours add 1 ul of Rnase A (10mg/ml) and incubate at 37C for 20 minutes.
Purify 1st strand cDNA products on qiagen PCR purification column, eluting with 50 ul water
[It is possible to run a 1% agarose gel at this point to assess cDNA quality.]
Klenow Labelling -(Random primers and klenow from Bioprime kit (Invitrogen))
Add the following;
Target cDNA 20 uL
[2.5x] Random primer/Reaction buffer mix 20 uL
Boil x 5 min - cool on ice.
Add, while on ice;
[10x] dNTP mix(low dC) 5 uL
1 mM Cy5-dCTP 3 uL
Klenow (high conc.) 1.5 uL
Incubate @ 37 C for between 2 and 4 hours
Stop reaction with 0.5 M EDTA (pH 8.0) x 5 uL.
Pass each labelling reaction down a sephadex G50 spin column.
I use Probequant columns [from Amersham] for this - each column loads 50 ul sample and 90% of the unincorporated nucleotide is removed. Reverse Transcription
Total RNA x uL (50 ug total)
Oligo (dT) (T15) (0.5ug/uL) (Promega) 8 uL (4 ug total)
dH2O RNase-free (Sigma) to 24 uL
Heat to 70 0C x 5 min. and snap cool on Ice x 1min
Prepare the following:
[5x] RT buffer (BRL) 10 uL
0.1M DTT (BRL) 5 uL
25 mM dNTPs (including dCTP) 2 uL
RNase inhibitor (BRL) 1 uL
dH2O RNase-free (Sigma) 6 uL
Add the above mix to the RNA/Oligo (dT) solution & mix.
Add 2 uL of M-MLV II (Superscript II, BRL) & incubate at 42C. Add further 1 ul after each 60 minutes.
After 4 hours add 1 ul of Rnase A (10mg/ml) and incubate at 37C for 20 minutes.
Purify 1st strand cDNA products on qiagen PCR purification column, eluting with 50 ul water
[It is possible to run a 1% agarose gel at this point to assess cDNA quality.]
Klenow Labelling -(Random primers and klenow from Bioprime kit (Invitrogen))
Add the following;
Target cDNA 20 uL
[2.5x] Random primer/Reaction buffer mix 20 uL
Boil x 5 min - cool on ice.
Add, while on ice;
[10x] dNTP mix(low dC) 5 uL
1 mM Cy3-dCTP 3 uL
Klenow (high conc.) 1.5 uL
Incubate @ 37 C for between 2 and 4 hours
Stop reaction with 0.5 M EDTA (pH 8.0) x 5 uL.
Pass each labelling reaction down a sephadex G50 spin column.
I use Probequant columns [from Amersham] for this - each column loads 50 ul sample and 90% of the unincorporated nucleotide is removed. Combine the purified Cy-3 & Cy-5 probes together
Add blocking agents;
Cot1 1ug/uL 10-20 uL
Poly(dA) 8ug/uL 2 uL
It is OK to miss out the Poly(dA) when using oligonucleotide arrays as there are no poly A or polyT tracts on the array.
Evaporate the combined sample to a volume of 12.5ul (or to dryness) in a speedvac
Resuspend sample in water 12.5ul
Add 37.5 uL of microarray hybridization solution (50% formamide, 5 x SSC, 0.1% SDS).
NB We find it easier to resuspend pellets in water so our hyb is made up at 1.3x concentrate
Incubate this target mixture at 85C for 5 minutes
Incubate labeling reaction at 42 C x 30 - 60 min prior to placing on array.
Place array slide into hybridisation cassette (Corning) and put 15ul of water in each end of chamber to ensure humidity. Seal chamber and place in oven at 42C for 30-60 minutes
Spin sample for 2 minutes at 13000rpm before applying to slide
Carefully add labeled mixture onto slide (avoid picking up any solid matter), add coverslip & cassette lid & hybridise at 48 C overnight.
Slide Washing
Place slides (in rack) in container with 1 L of Wash solutions A (2 x SSC)until cover slip falls off.
Transfer rapidly to container with 1L Wash solution B (0.1 x SSC, 0.1% SDS)& mix vigorously x 5mins
Transfer rapidly to container with 1L Wash solution C (0.1 x SSC)and mix vigorously 2mins
Do not expose any portion of the slides to the air!
Quickly move slides into 50mL Falcon tube USING FORCEPS & taking care not to touch surface.
Place slides in tube with labelled end in the bottom of the falcon tube (this means that any debris is spun away from the array, not over it).
Spin for 5 mins x 600rpm @ RT
Move slides to fresh, dry 50 mL tubes. Inspect slides immediately for liquid, and spin again if required (then move to another dry tube). Speed is vital at this stage, if liquid dries on the slide the quality of the array will suffer.
Scan array. 1.Turn on the scanner.
2.Double click on affymetrix scanner icon from desktop.
3.Wait until the scanner is configured and has warmed up.
4.Place the slide array side up on the platform at the front of the scanner, with the barcode end going into the scanner last.
5.You should be able to lay your slide onto the platform and slide it into the slot at the front up to about 3/4 of the way in.
6.From the run menu at the top of the software window select start scan, a new window will appear with the settings for the scanner, in the base file name box type the name for the file which you are about to scan.
7.From the dye drop down box select the dye required.
8.Set the gain at which to scan, a typical range would be from 30 to 50 this is dependent on the strength of signal on the slide.
9.After selecting the gain and laser and other settings press the set button for the changes to take effect.
10.On the right hand side of the window is a representation of the slide, move the hatched box to select the area of the slide to be scanned.
11.To start scanning click 'start scan'.
12.On the screen there is a small window called live image, select this and maximise it, as the scanner continues the image will be displayed.
13.The image will appear on screen in a false colour representation of the grayscale data, this set up so that intensity levels run from black to blue, green, yellow, red, white. White is at the limit of detection and indicate that the flouresence is saturating the reader
14.If the gain is incorrectly set whilst the scan is running you can click on the stop icon or select stop from the run menu, and then select start scan from the run menu or click on the traffic light icon, change the gain press the set button and start scan. When the scan has finished the image will be corrected and appear as normal, if you are satisfied with the scan then select save.
15.Once you have saved your image select start scan and change the dye and gain settings and start again.
16.Once you have finished and saved all the images you require go to the run menu and select eject slide.
Protocol Parameters Amplification;Extracted product; Amount of nucleic acid labeled;Label used;Amplification; Amplification;Label used;Amount of nucleic acid labeled; time;Quantity of label target used;Chamber type;temperature;Volume;
Protocol Hardware Scanning hardware
Protocol Software Default scanner software
Protocol Contact
Protocol Term Source REF mo mo mo mo mo mo
SDRF File E-MEXP-481.sdrf.txt
Term Source Name mo NCI_thesaurus atcc ncbitax ArrayExpress The MGED Ontology EFO mo
Term Source File http://mged.sourceforge.net/ontologies/MGEDontology.php ncithesaurus.obo.alt http://www.atcc.org/ 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/ http://mged.sourceforge.net/ontologies/MGEDontology.php
Term Source Version