Investigation Title Transcription profiling of human N-Acetyl L-cysteine induced differentiation of NHEK cells in vitro
Comment[Submitted Name] KTH H.Sapiens NAC induced differentiation of NHEK cells
Experimental Design time_series_design reference_design compound_treatment_design transcription profiling by array
Experimental Design Term Source REF mo mo mo EFO
Comment[ArrayExpressReleaseDate] 2005-11-30
Comment[AEMIAMESCORE] 3
Comment[ArrayExpressAccession] E-MEXP-500
Comment[MAGETAB TimeStamp_Version] 2010-08-11 15:20:10 Last Changed Rev: 13058
Experimental Factor Name time compound
Experimental Factor Type time compound_treatment_design
Experimental Factor Term Source REF
Person Last Name Lundeberg Parasassi Edlundh-Rose Greco Serafino Nilsson Bracci-Laudiero Kupershmidt Gustafsson Lundeberg Krasnowska Romano
Person First Name Thomas Tiziana Esther Giulia Annalucia Peter Luisa Ilya Anna Joakim Ewa Maria-Concetta
Person Mid Initials S C K
Person Email esther@biotech.kth.se
Person Phone +4655378343
Person Fax
Person Address Roslagstulssbacken 21
Person Affiliation Karolinska University Hospital, Sweden CNR, Italy Biotechnology CNR, Italy CNR, Italy Royal Institute of Technology CNR, Italy Silicon Genetics Royal Institute of Technology Royal Institute of Technology CNR, Italy Associazione Italiana Iniziativa Medicina Sociale
Person Roles submitter
Person Roles Term Source REF
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 2005-11-30
PubMed ID 16127296
Publication DOI 16127296
Publication Author List Esther Edlundh-Rose; Ilya Kupershmidt; Anna Gustafsson; Tiziana Parasassi; Annalucia Serafino; Luisa Bracci-Laudiero; Giulia Greco; Ewa Krasnowska; Maria-Concetta Romano; Thomas Lundeberg; Peter Nilsson; Joakim Lundeberg
Publication Title Gene expression analysis of human epidermal keratinocytes after N-acetyl L-cysteine treatment demonstrates cell cycle arrest and increased differentiation
Publication Status journal_article
Publication Status Term Source REF
Experiment Description Gene expression analysis of N-Acetyl L-cysteine induced differentiation of NHEK cells in vitro.
Protocol Name P-MEXP-15590 P-MEXP-15589 P-MEXP-15586 P-MEXP-15588 P-MEXP-5570 P-MEXP-5572 P-MEXP-10392 P-MEXP-15587
Protocol Type specified_biomaterial_action grow nucleic_acid_extraction pool labeling labeling hybridization feature_extraction
Protocol Description N-acetyl L-cysteine stock solution (20 mM in KGM) stored at 4 C and in the dark, was used within 1 week of preparation. Twenty-four hours after seeding, filter-sterilised NAC solution was added to the cell cultures to a final concentration of 2 mM Normal human epidermal keratinocytes, plated at a density of 8 x 103/cm2, were grown in KGM medium plus KGM Single Quots. Total RNA was extracted from cell cultures using TRIzol according to the manufacturer's instructions. Equal amounts of amplified RNA for all samples in the experiment were pooled before labelling was performed. Amplification protocol
A. Isolation of mRNA (according to Dynal's "RNA Direct™" protocol)
Wash oligodT beads (Dynal): Take 10 ml beads to each sample. Wash once with binding buffer. Add 10 ml binding.
Add 10 ml totRNA in RNasin buffer.
RNasin buffer:
78 ul RNase free H2O
20 ul 5 x 1st Strand Buffer (Invitrogen)
2 ul RNasin (Gibco)
Let bind for 5 min at room temp, on a "roller"
Remove supernatant and wash the beads: 2x Washing buffer B (without LiDS)
To elute mRNA: add 5 ml Rnase free water
Incubate at 65 C for 2min (mix a little once or twice)
Move supernatant, containing mRNA, to new tube
Repeat elution once (giving a total of 10 ml mRNA)
B. First strand cDNA synthesis (according to Life Technologies "SuperScript Choice System for cDNA Synthesis" protocol)
10 ml mRNA
2 ml biotinylated NotI-oligodT primer (0.5 mg/ml)
Incubate at 70 C for 10 min (reduce secondary structure in mRNA)
Cool quickly on ice and spin down
Add: 4 ml 5x first strand buffer
2 ml 0.1 M DTT
1 ml 10 mM dNTP mix
Vortex gently, spin down
Incubate at 42 C for 2 min
Add: 1 ml SuperScript II
Incubate at 42 C for 1 hour
Terminate reaction on ice
C. Second strand cDNA synthesis (Life technologies)
On ice, add to the above reaction (in right order):
91 ml RNase free water
30 ml 2nd strand buffer
3 ml 10 mM dNTP mix
1 ml E. Coli DNA ligase (10 U/ml)
4 ml E. Coli DNA polymerase I (10 U/ml)
1 ml Rnase H (2 U/ml)
Vortex gently
Incubate at 16 C for 2 hours (not above 16 )
Add: 3 ml T4 DNA polymerase
Incubate another 5 min at 16
Put on ice
Add: 10 ml 0.5 M EDTA (to terminate reaction)
Phenol extraction & EtOH precipitation:
Add: 150 ml phenol:chloroform:isoamylalchohol (25:24:1)
Vortex carefully
Centrifuge for 5 min at 12100 rpm
Transfer upper phase (140 ml) to new tube
Add: 70 ml 7.5 M NH4OAc
500 ml 96% EtOH (-20 C)
1 ml glycogen (20 mg/ml)
Vortex
Centrifuge immediately at 12100 rpm for 20 min, Remove supernatant
Wash pellet (hardly visible) with 500 ml 70% EtOH (-20 C)
Centrifuge 12100 rpm, 15 min
Remove supernatant, let pellet dry in room temp
Dissolve pellet in 40 ml 1xTE
D. Control step; PCR
To make sure you have cDNA; run PCR with housekeepning gene primers, e.g. GAPDH or actin.
E. Generation of 3´-tags
1. Remove primers (Clontech chromaspin TE-100 spin columns):
Add 2 mg glycogen to 40 ml cDNA sample from above
Run through Clontech TE-100 columns acording to manufacturer's instructions
2. Sonicate:
Level 9. (Inverted sonicator Sonifier ® B-12, Branson Sonic Power, if other
sonicator is used level and no. of cycles probably have to be optimized). Sonicate 16 times for 10 seconds. Each pause should be 15 sec or more, every other time on ice (sonication generates heat)
3. Bind to streptavidin beads (Dynal m-280)
Take 20 ml beads per sample
Wash beads twice with 40 ml binding/washing buffer
Remove buffer
Add: 40 ml binding/washing buffer
40 ml sonicated sample
Bind 3'-tags (biotinylated) by incubating at 37 C for 1 hour on "roller"
During incubation begin with step 5.
4. Repair fragments by T4 DNA pol blunt ending
Remove 80 ml buffer/sample from beads
Wash beads with 1 x T4 DNA pol buffer (2 x 40 ml)
Remove wash buffer
Add to beads: 3 ml 10 x T4 DNA pol buffer
1.5 ml BSA (1 mg/ml)
0.5 ml T4 DNA polymerase (3 U/ml)
3 ml dNTP mix (1 mM)
22 ml water
Incubate at 12 C for maximum 20 min, mix a few times to keep beads in solution
Remove repair mix from beads
Wash beads with "1 x 660 ligase buffer" (2 x 50 ml)
5. Ligation of adaptors
Mix in PCR tube: 2 ml Sima19 primer (278 pmol/ml)
2 ml Sima18 primer (278 pmol/ul)
3 ml "10 x 660 ligase buffer" (the buffer supplied with the
enzyme could probably work as well)
3 ml ATP (2 mM)
18.5 ml H2O
Anneal Sima19 and Sima18 by running in a PCR machine 50 C for 1 min, then a ramp to10 C over 1 hour.
Remove the wash buffer from the beads in step 4 and add the above.
Add 1,5 ml T4 DNA ligase (400 U/ml)
Incubate over night at cool room temperature (preferably down to 16 , but 20 works fine), on a "roller"
6. Cleave the 3'-tags from the beads (NotI)
Remove the ligation mixture from the beads.
Wash beads with 1 x NEB3 buffer (New England Biolabs)
Remove wash buffer
Add: 6 ml 10 x NEB3 buffer
6 ml BSA (1 mg/ml)
1 ml NotI (10 U/ml)
47 ml H2O
Incubate at 37 C for 2 hours
Transfer supernatant (with 3'-tags) to new tube
Heat inactivate enzyme at 70 C for 20 min
F. Amplify 3´-tags
PCR amplify 3'-tags using Sima19 and NotI-oligodT,
according to The Plant Journal (2001) 25(5), 585-591
Optimization of the numbers of cycles needs to be done before you run your final PCR. Take out 7 ml of sample every 5 cycles. Run on agarose gel. The optimal number of cycles is achieved appr 2 cycles before "saturation".
G. Labeling of PCR product (by asymmetric PCR)
The labeling protocol has been further optimized since the Plant-paper was published:
1. Remove primers (QIAquick column PCR purification from Qiagen):
Do this according to protocol, except:
- Wash twice with buffer PE
- Elute two times with 30 ml of buffer EB each time (total of 60 ml), let the column stand for 1 minute with the EB before centrifugation both times.
2. Check the concentration of PCR product
Dilute the sample 10 times, check the concentration using a spectrophotometer.
100 to 200 ng of PCR product (usually 4-5 ml) should be used in the labeling reaction.
3. Labeling through asymmetric PCR
Mix: x ml PCR product (100-200 ng)
5 ml AmpliTaq Gold buffer
4 ml MgCl2 (supplied with PE AmpliTaq Gold)
5 ml dNTP mix (see below)
3 ml Cy3-dCTP (1nmol/ml, we prefer the nucleotides supplied from
Perkin Elmer/NEN)
1 ml primer (Sima19, 50 pmol/ml)
0,6 ml AmpliTaq Gold
y ml water
Total: 50 ml
Cycle as follows:
95 C 12 min
95 C 30 sec ; 50 C 30 sec ; 72 C 10 min (20 cycles)
4 C
4. Remove primers (QIAquick column PCR purification from Qiagen):
As above, under point 1.
Speedvac samples until dry. Store away from the light.
Hybridize!!! Amplification protocol
A. Isolation of mRNA (according to Dynal's "RNA Direct™" protocol)
Wash oligodT beads (Dynal): Take 10 ml beads to each sample. Wash once with binding buffer. Add 10 ml binding.
Add 10 ml totRNA in RNasin buffer.
RNasin buffer:
78 ul RNase free H2O
20 ul 5 x 1st Strand Buffer (Invitrogen)
2 ul RNasin (Gibco)
Let bind for 5 min at room temp, on a "roller"
Remove supernatant and wash the beads: 2x Washing buffer B (without LiDS)
To elute mRNA: add 5 ml Rnase free water
Incubate at 65 C for 2min (mix a little once or twice)
Move supernatant, containing mRNA, to new tube
Repeat elution once (giving a total of 10 ml mRNA)
B. First strand cDNA synthesis (according to Life Technologies "SuperScript Choice System for cDNA Synthesis" protocol)
10 ml mRNA
2 ml biotinylated NotI-oligodT primer (0.5 mg/ml)
Incubate at 70 C for 10 min (reduce secondary structure in mRNA)
Cool quickly on ice and spin down
Add: 4 ml 5x first strand buffer
2 ml 0.1 M DTT
1 ml 10 mM dNTP mix
Vortex gently, spin down
Incubate at 42 C for 2 min
Add: 1 ml SuperScript II
Incubate at 42 C for 1 hour
Terminate reaction on ice
C. Second strand cDNA synthesis (Life technologies)
On ice, add to the above reaction (in right order):
91 ml RNase free water
30 ml 2nd strand buffer
3 ml 10 mM dNTP mix
1 ml E. Coli DNA ligase (10 U/ml)
4 ml E. Coli DNA polymerase I (10 U/ml)
1 ml Rnase H (2 U/ml)
Vortex gently
Incubate at 16 C for 2 hours (not above 16 )
Add: 3 ml T4 DNA polymerase
Incubate another 5 min at 16
Put on ice
Add: 10 ml 0.5 M EDTA (to terminate reaction)
Phenol extraction & EtOH precipitation:
Add: 150 ml phenol:chloroform:isoamylalchohol (25:24:1)
Vortex carefully
Centrifuge for 5 min at 12100 rpm
Transfer upper phase (140 ml) to new tube
Add: 70 ml 7.5 M NH4OAc
500 ml 96% EtOH (-20 C)
1 ml glycogen (20 mg/ml)
Vortex
Centrifuge immediately at 12100 rpm for 20 min, Remove supernatant
Wash pellet (hardly visible) with 500 ml 70% EtOH (-20 C)
Centrifuge 12100 rpm, 15 min
Remove supernatant, let pellet dry in room temp
Dissolve pellet in 40 ml 1xTE
D. Control step; PCR
To make sure you have cDNA; run PCR with housekeepning gene primers, e.g. GAPDH or actin.
E. Generation of 3´-tags
1. Remove primers (Clontech chromaspin TE-100 spin columns):
Add 2 mg glycogen to 40 ml cDNA sample from above
Run through Clontech TE-100 columns acording to manufacturer's instructions
2. Sonicate:
Level 9. (Inverted sonicator Sonifier ® B-12, Branson Sonic Power, if other
sonicator is used level and no. of cycles probably have to be optimized). Sonicate 16 times for 10 seconds. Each pause should be 15 sec or more, every other time on ice (sonication generates heat)
3. Bind to streptavidin beads (Dynal m-280)
Take 20 ml beads per sample
Wash beads twice with 40 ml binding/washing buffer
Remove buffer
Add: 40 ml binding/washing buffer
40 ml sonicated sample
Bind 3'-tags (biotinylated) by incubating at 37 C for 1 hour on "roller"
During incubation begin with step 5.
4. Repair fragments by T4 DNA pol blunt ending
Remove 80 ml buffer/sample from beads
Wash beads with 1 x T4 DNA pol buffer (2 x 40 ml)
Remove wash buffer
Add to beads: 3 ml 10 x T4 DNA pol buffer
1.5 ml BSA (1 mg/ml)
0.5 ml T4 DNA polymerase (3 U/ml)
3 ml dNTP mix (1 mM)
22 ml water
Incubate at 12 C for maximum 20 min, mix a few times to keep beads in solution
Remove repair mix from beads
Wash beads with "1 x 660 ligase buffer" (2 x 50 ml)
5. Ligation of adaptors
Mix in PCR tube: 2 ml Sima19 primer (278 pmol/ml)
2 ml Sima18 primer (278 pmol/ul)
3 ml "10 x 660 ligase buffer" (the buffer supplied with the
enzyme could probably work as well)
3 ml ATP (2 mM)
18.5 ml H2O
Anneal Sima19 and Sima18 by running in a PCR machine 50 C for 1 min, then a ramp to10 C over 1 hour.
Remove the wash buffer from the beads in step 4 and add the above.
Add 1,5 ml T4 DNA ligase (400 U/ml)
Incubate over night at cool room temperature (preferably down to 16 , but 20 works fine), on a "roller"
6. Cleave the 3'-tags from the beads (NotI)
Remove the ligation mixture from the beads.
Wash beads with 1 x NEB3 buffer (New England Biolabs)
Remove wash buffer
Add: 6 ml 10 x NEB3 buffer
6 ml BSA (1 mg/ml)
1 ml NotI (10 U/ml)
47 ml H2O
Incubate at 37 C for 2 hours
Transfer supernatant (with 3'-tags) to new tube
Heat inactivate enzyme at 70 C for 20 min
F. Amplify 3´-tags
PCR amplify 3'-tags using Sima19 and NotI-oligodT,
according to The Plant Journal (2001) 25(5), 585-591
Optimization of the numbers of cycles needs to be done before you run your final PCR. Take out 7 ml of sample every 5 cycles. Run on agarose gel. The optimal number of cycles is achieved appr 2 cycles before "saturation".
G. Labeling of PCR product (by asymmetric PCR)
The labeling protocol has been further optimized since the Plant-paper was published:
1. Remove primers (QIAquick column PCR purification from Qiagen):
Do this according to protocol, except:
- Wash twice with buffer PE
- Elute two times with 30 ml of buffer EB each time (total of 60 ml), let the column stand for 1 minute with the EB before centrifugation both times.
2. Check the concentration of PCR product
Dilute the sample 10 times, check the concentration using a spectrophotometer.
100 to 200 ng of PCR product (usually 4-5 ml) should be used in the labeling reaction.
3. Labeling through asymmetric PCR
Mix: x ml PCR product (100-200 ng)
5 ml AmpliTaq Gold buffer
4 ml MgCl2 (supplied with PE AmpliTaq Gold)
5 ml dNTP mix (see below)
3 ml Cy3-dCTP (1nmol/ml, we prefer the nucleotides supplied from
Perkin Elmer/NEN)
1 ml primer (Sima19, 50 pmol/ml)
0,6 ml AmpliTaq Gold
y ml water
Total: 50 ml
Cycle as follows:
95 C 12 min
95 C 30 sec ; 50 C 30 sec ; 72 C 10 min (20 cycles)
4 C
4. Remove primers (QIAquick column PCR purification from Qiagen):
As above, under point 1.
Speedvac samples until dry. Store away from the light.
Hybridize!!! Hybridisation protocol for 3' tag amplified samples
Prehybridisation of chip:
1. Prehybridisation solution
2g BSA
148 ml H2O
50 ml 20 x SSC
2 ml 10% SDS
Filter 50 ml of solution through a 0,45um filter using a syringe, into a washing chamber with screw top lid. Preheat to 42C in a water bath for approx. 30 min.
2. Put the microarrays in the chamber and shake. Incubate at 42C in the water bath for 45 min.
3. Wash the arrays by dipping and shaking in H2O 4-5 times. Dip in isopropanol and centrifuge immediately in a chip centrifuge. Use the array within an hour.
Hybridisation
Make sure to work in the dark, as samples are sensitive to light.
1. Hybridisation buffer
500 ul H2O
240 ul formamide
250 ul 20 x SSC
10 ul 10% SDS
Dissolve samples in a total of 60 ul hybridisation buffer. Add 2.5 ul human cot-1 DNA (10 ug/ul) and 2.5 ul polyA DNA (20ug/ul).
2. Put 10 ul H2O in the humidity wells in the hybridisation chamber. Place the array, face up, in the chamber. Place the coverslip (lifterslip) with the white edges facing down, on the array.
3. Denaturate the samples by incubating 95C for 3 min.
4. Apply the sample by pipetting along the bottom edge of the coverslip, let the capillary action draw the sample upwards, to cover the whole array.
5. Close the chamber and cover in foil. Incubate at 42C in a water bath for 16-20 hours.
6. Wash the arrays as follows:
wash 1
2 x SSC
0.1% SDS
42C
Carefully remove the coverslip and incubate arrays for 4 min in preheated wash buffer on a shaker.
wash 2
0.1 x SSC
0.1% SDS
RT
Incubate arrays for 4 min in preheated wash buffer on a shaker.
wash 3
0.1 x SSC
RT
Incubate arrays for 1 min in preheated wash buffer on a shaker. Repeat 4 times. It is important that all SDS be removed.
Centrifuge immediately and keep out of the light.
7. Scan.
The scanner that was used was the Agilent G2565BA DNA Microarray scanner.
The slides were scanned at 10 um resolution immediately after washing.
Obtained images were rotated and the mirror image created using the provided software.
Protocol Parameters start time;min temperature;max temperature;stop time; Amplification;Extracted product; Label used;Amount of nucleic acid labeled;Amplification; Amount of nucleic acid labeled;Label used;Amplification; temperature;Quantity of label target used;Chamber type;
Protocol Hardware G2565BA DNA microarray scanner [Agilent]
Protocol Software Feature Extraction Software [Agilent]
Protocol Contact
Protocol Term Source REF mo
SDRF File E-MEXP-500.sdrf.txt
Term Source Name ncbitax mo ArrayExpress The MGED Ontology mo EFO
Term Source File http://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/arrayexpress http://mged.sourceforge.net/ontologies/MGEDontology.php http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/efo/
Term Source Version