Affymetrix GeneChip Murine Genome U74Av2 [MG_U74Av2]

Comprehensive comparison of six microarray technologies

rawData

scan

processedDataMatrix

Biological comparison of gene expression profiles of adult male whole Muta™Mouse lung  with its immortalized 100% confluent epithelial lung cell line counterpart. White, P.A.,et al. 2003.  Development and characterization of an epithelial cell line from Muta™Mouse lung.  Environ Mol Mutagen 42,3 pgs 166-184

Transcription profiling of adult male whole MutaMouse lung with its immortalized 100% confluent epithelial lung cell line counterpart (Affymetrix)

Biological comparison of gene expression profiles of adult male whole MutaÂMouse lung  with its immortalized 100% confluent epithelial lung cell line counterpart. White, P.A.,et al. 2003.  Development and characterization of an epithelial cell line from MutaÂMouse lung.  Environ Mol Mutagen 42,3 pgs 166-184

Biological comparison of gene expression profiles of adult male whole Muta™Mouse lung  with its immortalized 100% confluent epithelial lung cell line counterpart. White, P.A.,et al. 2003.  Development and characterization of an epithelial cell line from Muta™Mouse lung.  Environ Mol Mutagen 42,3 pgs 166-184


Affymetrix:Protocol:Hybridization-EukGE-WS2v4

Title: Fluidics Station Protocol. Description:

Affymetrix:Protocol:Percentile

Title: Affymetrix CEL Analysis (Percentile). Description:

P-MEXP-3811

Muta™ Mouse lung epithelial cells (FE1) were cultured in a 1:1 mixture of DMEM:F12 nutrient mixture supplemented with 2% v/v FBS, 2 mM glutamine, 100 U/ml penicillin G, 100  g/ml streptomycin sulphate, and 1 ng/ml murine epidermal growth factor (Invitrogen life technologies, Burlington, ON, CA)  (White et al., 2003).  All incubations were carried out at 37°C, 95% humidity, and 5% CO2.  to 100% confluence (5-10 days post confluence)

P-MEXP-3812

Transgenic mouse strain 40.6 (i.e., Muta™Mouse) was bred locally and maintained under conditions approved by the Health Canada Animal Care Committee. Strain information, including the protocols employed for the insertion of the _gt10lacZ vector, can be found in Gossen et al. [1989]. Animals were euthanized by cervical dislocation. Lungs were perfused  with Phosphate buffered saline prior to lung removal.  

P-MEXP-3813

Trizol+Qiagen RNA Isolation Protocol for Cell Culture Sample LBERNDT 2003-12-15 TRIZOL RNA ISOLATION for Cell Culture  1. Lyse cells directly by adding 0.3-0.4 ml of TRIzol LS reagent to a 3.5 cm diameter dish.     (1.5 ml in 15 cm plate). Pass the cell lysate several times through pipette.  2. Incubate samples for 5 minutes at 15-30 C.  3. Add 0.2 ml per 0.75 ml TRIzol LS reagent of chloroform. Shake tubes vigorously by hand for      15 seconds and incubate for 2-15 min. at 15-30 C.  4.  Centrifuge the samples at no more than 12,000 xg for 10 min. at 2-8 C.  The RNA precipitate      should form a gel like pellet on the side and bottom of tube.  5. Remove the supernatant.  Was the RNA pellet once with 75% EtOH, adding at least 1 ml of      75% EtOH per 0.75 ml of TRIzol reagent Mix the sample by vortexing and centrifuge at no      more than 7,500 x g for 5 min. at 2-8 C.  6. Dry (briefly) the RNA pellet (air dry).  Dissolve the RNA in 100 ul  Rnase free water and      incubate for 10 min. at 55-60 C.   RNA cleanup with Qiagen RNeasy mini kit1 ADD 10 ìl  -mercaptoethanol to 1 ml RTL buffer. Dilute RPE buffer with 4 vol of EtOH (x5 dilution) 2 ADJUST VOLUME of sample to 100 ìl 3 ADD 350 ìl RLT. Mix 4 ADD 250 ìl 100% EtOH. Vortex 5 TRANSFER (vol=700 ìl) to spin column 6 SPIN at 8000 g, 15 sec 7 DISCARD flow-through 8 TRANSFER to new collection tube 9 ADD 500 ìl RPE, SPIN at 8000 g, 15 sec, discard flow-through 10 ADD 500 ìl RPE, SPIN at 8000 g, 15 sec, discard flow-through 11 SPIN for 2 min at max. speed 12 TRANSFER to new tube 13 ADD 30 ìl ddH2O. Leave to stand for 1 min. 14 SPIN at 8000 g for 1 min. DO NOT REPEAT. 15 Quantify by A260/280 16      Measure Quality using Agilent Bioanalyzer.       

P-MEXP-3814

Trizol+Qiagen modified RNA Extraction Protocol for Tissue Samples LBERNDT 2003-12-15 TRIZOL RNA ISOLATION (JHS)  I. Homogenisation of tissues Note: 5-10 mg of tissue provides enough RNA for 1 chip  However, with the following protocol, you need a minimum of 25 mg tissue, preferably 50-100 mg. Any water used in the protocol (including water to make 70% EtOH) should be rnase-free.  1. Remove samples (in Rnalater) from -20 C freezer and let thaw.  2. CLEAN HOMOGENISER. Rinse in ddH2O and rinsing in 70% EtOH  3. ADD TRIzol. Add 1 ml TRIzol per 50-100 mg tissue TRIzol is nasty on inhalation and contact. Contains phenol and guanidine isothiocyanate.  Use gloves and eye protection. If multiple samples are being done, use in fume hood  4. HOMOGENISE. Full power for 15 sec.  5. RINSE HOMOGENISER. Use ddH2O between samples and rinse with 70% EtOH when finished.   6. Following addition of TRIzol, store at r.t. Transfer to eddendorf if <1 ml  Phase separation  1. INCUBATE homogenised samples for 5 mins at r.t. Check no lumps of tissue are present. If they are,      repeat homogenisation  2. ADD CHLOROFORM (store room cupboard) (no additives such as isoamyl alcohol - no phenol          needed due to TRIzol). Use 0.2 ml per 1 ml of TRIzol. Cap samples  3. SHAKE by hand for 15 sec. Incubate at r.t. for 2-3 mins  4. SPIN at 12000g for 15 mins at 4ºC. Spin with no cap if  using 13 ml tubes. Sorvall located in ice room  5. REMOVE upper aqueous phase   RNA precipitation  1. ADD 2-propanol. Use 0.5 ml per 1 ml of TRIzol used in the initial homogenisation.  2. INCUBATE at r.t. for 10 min  3. SPIN at 12000g for 10 min at 4ºC  4. REMOVE supernatent  5. ADD 1 ml 75% EtOH  6. VORTEX  7. SPIN at 7500 g for 5 min at 4ºC  8. AIR DRY pellet for 5-10 mins   9. REDISSOLVE in 1 ul ddH2O per 1 mg tissue (100ul).   10. put in 55 C water bath for 10 minutes. If pellet has not completely redissolved, add another         100 ul of ddH2O and incubate another 5 minutes. (Remember to split sample before proceeding         to qiagen column.      RNeasy Mini Protocol for RNA clean up:      1. Adjust The sample volume to 100 µl with Rnase-free water and add 350 µl of  Lysis Buffer RLT          Mix the  thoroughly.  2. Add 250 ul of 100% EtOH  and mix thoroughly. Transfer to a Qiagen  spin  column.  3.  Centrifuge for 15 seconds at >10,000 rpm (8 000 x g).  Discard flow through.   4. Transfer into a new 2 ml collection tube and add 500 μl Buffer RPE. Centrifuge for 15         seconds at > 10,000 rpm. Discard the flow through.  5.  Add another 500 μl RPE to the column and centrifuge for for 15  seconds at > 10,000 rpm.                 Discard the flow through.   6. Spin for 2 minutes at max speed to dry the silica-gel membrane.       7. To elute, transfer the columns to  new 1.5 ml collection tubes and add 50 ul RNAse-free         water, pipetting directly onto the membrane and leave stand for 1 minute.  8.  Centrifuge  for 1 minute at 10,000 rpm.  Do not repeat.  9. Measure A260/280 ratio on UV spec. and proceed with 28s/18s meaurements on bioanalyzer.        

P-MEXP-3815

Total RNA from 11 mouse cell lines was supplied by Stratagene. Catalog # 740100

P-MEXP-3827

P-MEXP-3891

We used dChip (PM only) to normalize within each tissue type (within FE1, within ref, within Lung). Li, C. & Hung Wong, W. Genome Biol 2, RESEARCH0032 (2001). Data was then imported into GeneSpring 6.1 (Silicon Genetics). Each chip was normalized to the 50th percentile taken from that chip using only measurements flagged present or marginal, and only measurements with raw signals of at least 10. Then, each chip was normalized per gene to the reference samples (3 chips that have Stratagene Universal mouse reference RNA on them) – that divided each gene in the sample by the median of that gene’s measurement in the control sample. Numbers are the normalized ratios to reference (not logged).

Transcription profiling of adult male whole MutaMouse lung with its immortalized 100% confluent epithelial lung cell line counterpart (Affymetrix)

Submitter Supplied Information

Samples