title: 3000054261

description:
I. Labelling Protocol (VIB-MAF Cy3 Labeling v1 and  VIB-MAF Cy5 Labeling v1)
[1] RNA amplification by in vitro transcription
This protocol has been published in L. G. Puskás, Á. Zvara, L. Hackler Jr., and P. Van Hummelen (2002). RNA amplification results in reproducible microarray data with slight ratio bias. Biotechniques (2002) vol. 32, no. 6 p.1330-1341.
1.1 Reagents:
------------
(1) RNase Out 40U/ul (Invitrogen, 5000U, Cat. No. 10777-019),
(2) HT7T primer, HPLC purified (Eurogentec, sequence: 5 - GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGGTTTTTTTTTT TTTTTTTTTTTTTT-3 ),
(3) Trehalose 1.7 M (Sigma, Cat. No. T-5251),
(4) 5 x first strand buffer (SuperScript II RT, Invitrogen, Cat. No. 18064-014),
(5) 0.1 M DTT (SuperScript II RT, Invitrogen, Cat. No. 18064-014),
(6) 10 mM dNTP-mix (Amersham Pharmacia Biotech, 100 mM, Cat. No. 27-2035- 01),
(7) SuperScript II RT 200U/ul (Invitrogen, 10000U, Cat. No. 18064-014),
(8) 5 x second strand buffer: 100 mM Tris-HCl (pH 6.9) (Invitrogen, Cat. No. 15714- 025 (Tris) and 1027 (HCl)), 450 mM KCl (Sigma, Cat. No. P-9541), 23 mM MgCl2 (Sigma, Cat. No. M 8266), 0.75 mM Beta-NAD+ (Sigma, Cat. No. N- 1636), 50 mM (NH4)2SO4 (Invitrogen, Cat. No. 15501-018)),
(9) E. coli DNA Ligase 10U/ul (Invitrogen, 100U, Cat. No. 18052-019),
(10) E. coli DNA Polymerase I 10U/ul (Invitrogen, 1000U, Cat. No. 18010-025),
(11) E. coli RNase H 2U/ul (Invitrogen, 120U, Cat. No. 18021-071),
(12) DEPC-water (Invitrogen, Cat. No. 10977-015),
(13) Ethanol Absolut (VWR, Cat. No. 1.08543.0250),
(14) QIAquick PCR Purification Kit (Qiagen, Cat. No 28106),
(15) NaAc 3M, pH 5.0 (Sigma, Cat. No. S-2889),
(16) AmpliScribeTM T7 Transcription Kit (Epicentre Technologies, Cat. No. AS3107),
(17) RNeasy Mini Kit (Qiagen, Cat. No 74106).
1.2 Protocol First Strand Synthesis:
-----------------------------------
(1) Mix 5 ug of total RNA (3 ul) with 1 ul of RNase Out 40U/ul.
(2) Add 1 ul of (HT7T) primer 200 ng/ul and 6 ul of 1.7 M Trehalose heated to 42C.
(3) Put on 75C for 5 minutes, chill on ice and spin down.
(4) Add 4 ul of 5 x first strand buffer, 2 ul of 0.1 M DTT, 1 ul of 10mM dNTP-mix, 1 ul of 1.7 M Trehalose and 1 ul of SuperScript II RT.
(5) Incubate at 37C for 5 minutes, at 45C for 5 minutes and then make 10 cycles of 60C for 2 minutes and 55C for 2 minutes. Go to 65C for 10 minutes and then go to 4C or chill on ice.
1.3 Protocol Second Strand Synthesis:
------------------------------------
(1) Add 33.4 ul of 5 x second strand buffer, 3.4 ul of 10 mM dNTP-mix, 1 ul of E. coli DNA Ligase (10U/ul), 4 ul of E. coli DNA Polymerase I (10U/ul), 1 ul of E. coli RNase H (2U/ul) and 103.8 ul of DEPC-water to the 20 ul of first strand mix.
(2) Incubate at 16C for 3 hours, at 65C for 10 minutes and then go to 4C or chill on ice.
(3) Purification with QIAquick PCR Purification Kit according to the manufacturers guidelines.
1.4 Protocol for In Vitro Transcription (IVT):
---------------------------------------------
(1) Add 2 ul of 10 x T7 reaction buffer (heated to 42C), 6 ul of 25 mM rNTP-mix, 2 ul of 100 mM DTT and 2 ul of AmpliScribe T7 enzyme solution to the DNA from second strand synthesis.
(2) Incubate at 37C for 3 hours, at 65C for 10 minutes and then go to 4C or chill on ice.
(3) Purification with QIAgen RNeasy Mini Kit according to the manufacturers guidelines.
(4) Check RNA concentration by OD. We recommend a 1/10 dilution in water or no dilution when using Nanodrop. Check A260/A280 ratio and determine the RNA concentration (1 OD = 40 ng/ul).
[2] Labeling
2.1 Reagents:
------------
(1) RNase Out 40U/ul (Invitrogen, 5000U, Cat. No. 10777-019),
(2) Random nonamers N9 1 ug/ul (Genset, 1862270),
(3) 5 x first strand buffer (SuperScript II RT, Invitrogen, Cat. No. 18064-014),
(4) 0.1 M DTT (SuperScript II RT, Invitrogen, Cat. No. 18064-014),
(5) 10 mM dNTP/2 mM dCTP (Amersham Pharmacia Biotech, 100 mM, Cat. No. 272050/60/70/80),
(6) CyTM-3-dCTP and CyTM-5-dCTP for microarrays (Amersham Pharmacia Biotech, 25 nmol, Cat. No. PA 53021 and PA 55021),
(7) SuperScript II RT 200U/ul (Invitrogen, 10000U, Cat. No. 18064-014),
(8) 2.5 M NaOH (Sigma, Cat. No. S-0899),
(9) MOPS (3-(N-Morpholino)propanesulfonic acid) (Sigma, Cat. No. M-8899),
(10) NaAc 3M, pH 5.0 (Sigma, Cat. No. S-2889),
(11) QIAquick PCR Purification Kit (Qiagen, Cat. No 28106).

2.2 Cy3 labeling Protocol:
----------------------
(1) Mix 5 ug of RNA from IVT (8 ul), 0.5 ul of RNase Out 40U/ul, 5 ul spikes (not for Arabidopsis thaliana) and 2 ul of random nonamers N9 1 ug/ul.
(2) Denaturate at 70C for 10 minutes, chill on ice and add the following components: 4 ul of first strand buffer, 1 ul of 10 mM dNTP/2 mM dCTP, 2 ul of 0.1 M DTT, 1.5 ul Cy3 dCTP and 1 ul of SuperScript II RT 200U/ul.
(3) Incubate at 42C for 2.5 hours and then go to 4C or chill on ice.
(4) Denaturate the RNA template by adding 2 ul of 2.5 M NaOH, incubating at 37C for exactly 15 minutes, adding 10 ul of 2 M MOPS and putting on ice.
(5) Purification with QIAquick PCR Purification Kit according to manufacturers guidelines.

2.3 Cy5 Labeling Protocol:
----------------------
(1) Mix 5 ug of RNA from IVT (8 ul), 0.5 ul of RNase Out 40U/ul, 5 ul spikes (not for Arabidopsis thaliana) and 2 ul of random nonamers N9 1 ug/ul.
(2) Denaturate at 70C for 10 minutes, chill on ice and add the following components: 4 ul of first strand buffer, 1 ul of 10 mM dNTP/2 mM dCTP, 2 ul of 0.1 M DTT, 1.5 ul of Cy5 dCTP and 1 ul of SuperScript II RT 200U/ul.
(3) Incubate at 42C for 2.5 hours and then go to 4C or chill on ice.
(4) Denaturate the RNA template by adding 2 ul of 2.5 M NaOH, incubating at 37C for exactly 15 minutes, adding 10 ul of 2 M MOPS and putting on ice.
(5) Purification with QIAquick PCR Purification Kit according to manufacturers guidelines.

II. Hybridisation Protocol (VIB-MAF Automatic hybridization v1 using Automatic Slide Processor (ASP))
A. Reagents:
-----------
(1) PolydT35 1 ug/ul (Genset),
(2) Mouse COT-1 DNA (R) 1 mg/ml (Invitrogen, 500 ug, Cat. No. 18440-016),
(3) Human COT-1 DNA (R)1 mg/ml (Invitrogen, 500 ug, Cat. No. 15279-011),
(4) Microarray hybridization buffer (Amersham Pharmacia Biotech, Cat. No. RPK0325),
(5) Formamide (Sigma, Cat. No. F-9037). Use Mouse COT DNA on Mouse slides, Human COT DNA on Human slides and no COT on Arabidopsis slides.
B. Protocol for automatic hybridization with ASP:
------------------------------------------------
(1) Make 210 ul hybridization mixture per slide with 25% probe, 25% buffer and 50% formamide.
(2) Dissolve the Cy3 and Cy5 sample together in 12 ul of polydT35, 30 ul of Mouse or Human or no (Arabidopsis) COT, water to make a total volume of 52.5 ul, 52.5 ul of Microarray 4x hybridization buffer and 105 ul formamide.
(3) Denature at 96C for 3 minutes.
(4) Keep on ice until hybridization.
(5) Inject samples in ASP.
III. Scanning Protocol (VIB-MAF scanning v1)
A. Equipment:
------------
(1) Generation III Scanner, Amersham Biosciences
(2) Scanner software: Default scanner software
B. Scanning Protocol:
--------------------
(1) Before scanning, adjust scanning parameters to: Full Slide, Cy5 PMT=550V, Cy3 PMT=550V
Keywords = Thyroid
Keywords = Adenosine Receptor 2a
Lot batch = type 7 star Amersham ; lot 32 ; RPK 2331 ; Printed 2-May-2002


title: 24 hr B-chip 6-13-02

status: Public on Oct 29 2003

type: RNA

city: Baltimore

first: Jerry

middle: M.

last: Wright

state: MD

address: 725 N. Wolfe St

department: Physiology

country: USA

institute: Johns Hopkins Medical Institutions

description: IB3-1 (deltaF508/W1282X) bronchial epithelial cells were cultured in T75 flasks with gentamicin-free LHC-8 medium.  After reaching 80% confluence cells were treated with 1 mM PBA for 24 hr.  A T75 flask of confluent IB3-1 cells was rinsed twice with ice cold Hank’s buffer then scraped into 3ml of ice cold TRIzol (Gibco BRL) then rinsed with 3 ml ice cold TRIzol and the mRNA was isolated according to the TRIzol protocol.  Array images were assessed by eye to confirm scanner alignment and the absence of significant bubbles or scratches.  Chip scanning and expression analysis was performed using Microarray Analysis Suite 5.0 software and scaled to an average probe set intensity of 150.

molecule: total RNA

organism: Homo sapiens

source_name: IB3-1 bronchial epithelial cell line


title: 3000002695

status: Public on Nov 25 2003

city: Leuven

first: Rekin's

last: Janky

address: Herestraat 49 Box 816

department: Nucleomics Core

country: Belgium

institute: VIB

description:
I. Labelling Protocol (VIB-MAF Cy3 Labeling v1 and  VIB-MAF Cy5 Labeling v1)
[1] RNA amplification by in vitro transcription
This protocol has been published in L. G. Puskás, Á. Zvara, L. Hackler Jr., and P. Van Hummelen (2002). RNA amplification results in reproducible microarray data with slight ratio bias. Biotechniques (2002) vol. 32, no. 6 p.1330-1341.
1.1 Reagents:
------------
(1) RNase Out 40U/ul (Invitrogen, 5000U, Cat. No. 10777-019),
(2) HT7T primer, HPLC purified (Eurogentec, sequence: 5 - GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGGTTTTTTTTTT TTTTTTTTTTTTTT-3 ),
(3) Trehalose 1.7 M (Sigma, Cat. No. T-5251),
(4) 5 x first strand buffer (SuperScript II RT, Invitrogen, Cat. No. 18064-014),
(5) 0.1 M DTT (SuperScript II RT, Invitrogen, Cat. No. 18064-014),
(6) 10 mM dNTP-mix (Amersham Pharmacia Biotech, 100 mM, Cat. No. 27-2035- 01),
(7) SuperScript II RT 200U/ul (Invitrogen, 10000U, Cat. No. 18064-014),
(8) 5 x second strand buffer: 100 mM Tris-HCl (pH 6.9) (Invitrogen, Cat. No. 15714- 025 (Tris) and 1027 (HCl)), 450 mM KCl (Sigma, Cat. No. P-9541), 23 mM MgCl2 (Sigma, Cat. No. M 8266), 0.75 mM Beta-NAD+ (Sigma, Cat. No. N- 1636), 50 mM (NH4)2SO4 (Invitrogen, Cat. No. 15501-018)),
(9) E. coli DNA Ligase 10U/ul (Invitrogen, 100U, Cat. No. 18052-019),
(10) E. coli DNA Polymerase I 10U/ul (Invitrogen, 1000U, Cat. No. 18010-025),
(11) E. coli RNase H 2U/ul (Invitrogen, 120U, Cat. No. 18021-071),
(12) DEPC-water (Invitrogen, Cat. No. 10977-015),
(13) Ethanol Absolut (VWR, Cat. No. 1.08543.0250),
(14) QIAquick PCR Purification Kit (Qiagen, Cat. No 28106),
(15) NaAc 3M, pH 5.0 (Sigma, Cat. No. S-2889),
(16) AmpliScribeTM T7 Transcription Kit (Epicentre Technologies, Cat. No. AS3107),
(17) RNeasy Mini Kit (Qiagen, Cat. No 74106).
1.2 Protocol First Strand Synthesis:
-----------------------------------
(1) Mix 5 ug of total RNA (3 ul) with 1 ul of RNase Out 40U/ul.
(2) Add 1 ul of (HT7T) primer 200 ng/ul and 6 ul of 1.7 M Trehalose heated to 42C.
(3) Put on 75C for 5 minutes, chill on ice and spin down.
(4) Add 4 ul of 5 x first strand buffer, 2 ul of 0.1 M DTT, 1 ul of 10mM dNTP-mix, 1 ul of 1.7 M Trehalose and 1 ul of SuperScript II RT.
(5) Incubate at 37C for 5 minutes, at 45C for 5 minutes and then make 10 cycles of 60C for 2 minutes and 55C for 2 minutes. Go to 65C for 10 minutes and then go to 4C or chill on ice.
1.3 Protocol Second Strand Synthesis:
------------------------------------
(1) Add 33.4 ul of 5 x second strand buffer, 3.4 ul of 10 mM dNTP-mix, 1 ul of E. coli DNA Ligase (10U/ul), 4 ul of E. coli DNA Polymerase I (10U/ul), 1 ul of E. coli RNase H (2U/ul) and 103.8 ul of DEPC-water to the 20 ul of first strand mix.
(2) Incubate at 16C for 3 hours, at 65C for 10 minutes and then go to 4C or chill on ice.
(3) Purification with QIAquick PCR Purification Kit according to the manufacturers guidelines.
1.4 Protocol for In Vitro Transcription (IVT):
---------------------------------------------
(1) Add 2 ul of 10 x T7 reaction buffer (heated to 42C), 6 ul of 25 mM rNTP-mix, 2 ul of 100 mM DTT and 2 ul of AmpliScribe T7 enzyme solution to the DNA from second strand synthesis.
(2) Incubate at 37C for 3 hours, at 65C for 10 minutes and then go to 4C or chill on ice.
(3) Purification with QIAgen RNeasy Mini Kit according to the manufacturers guidelines.
(4) Check RNA concentration by OD. We recommend a 1/10 dilution in water or no dilution when using Nanodrop. Check A260/A280 ratio and determine the RNA concentration (1 OD = 40 ng/ul).
[2] Labeling
2.1 Reagents:
------------
(1) RNase Out 40U/ul (Invitrogen, 5000U, Cat. No. 10777-019),
(2) Random nonamers N9 1 ug/ul (Genset, 1862270),
(3) 5 x first strand buffer (SuperScript II RT, Invitrogen, Cat. No. 18064-014),
(4) 0.1 M DTT (SuperScript II RT, Invitrogen, Cat. No. 18064-014),
(5) 10 mM dNTP/2 mM dCTP (Amersham Pharmacia Biotech, 100 mM, Cat. No. 272050/60/70/80),
(6) CyTM-3-dCTP and CyTM-5-dCTP for microarrays (Amersham Pharmacia Biotech, 25 nmol, Cat. No. PA 53021 and PA 55021),
(7) SuperScript II RT 200U/ul (Invitrogen, 10000U, Cat. No. 18064-014),
(8) 2.5 M NaOH (Sigma, Cat. No. S-0899),
(9) MOPS (3-(N-Morpholino)propanesulfonic acid) (Sigma, Cat. No. M-8899),
(10) NaAc 3M, pH 5.0 (Sigma, Cat. No. S-2889),
(11) QIAquick PCR Purification Kit (Qiagen, Cat. No 28106).

2.2 Cy3 labeling Protocol:
----------------------
(1) Mix 5 ug of RNA from IVT (8 ul), 0.5 ul of RNase Out 40U/ul, 5 ul spikes (not for Arabidopsis thaliana) and 2 ul of random nonamers N9 1 ug/ul.
(2) Denaturate at 70C for 10 minutes, chill on ice and add the following components: 4 ul of first strand buffer, 1 ul of 10 mM dNTP/2 mM dCTP, 2 ul of 0.1 M DTT, 1.5 ul Cy3 dCTP and 1 ul of SuperScript II RT 200U/ul.
(3) Incubate at 42C for 2.5 hours and then go to 4C or chill on ice.
(4) Denaturate the RNA template by adding 2 ul of 2.5 M NaOH, incubating at 37C for exactly 15 minutes, adding 10 ul of 2 M MOPS and putting on ice.
(5) Purification with QIAquick PCR Purification Kit according to manufacturers guidelines.

2.3 Cy5 Labeling Protocol:
----------------------
(1) Mix 5 ug of RNA from IVT (8 ul), 0.5 ul of RNase Out 40U/ul, 5 ul spikes (not for Arabidopsis thaliana) and 2 ul of random nonamers N9 1 ug/ul.
(2) Denaturate at 70C for 10 minutes, chill on ice and add the following components: 4 ul of first strand buffer, 1 ul of 10 mM dNTP/2 mM dCTP, 2 ul of 0.1 M DTT, 1.5 ul of Cy5 dCTP and 1 ul of SuperScript II RT 200U/ul.
(3) Incubate at 42C for 2.5 hours and then go to 4C or chill on ice.
(4) Denaturate the RNA template by adding 2 ul of 2.5 M NaOH, incubating at 37C for exactly 15 minutes, adding 10 ul of 2 M MOPS and putting on ice.
(5) Purification with QIAquick PCR Purification Kit according to manufacturers guidelines.

II. Hybridisation Protocol (VIB-MAF Automatic hybridization v1 using Automatic Slide Processor (ASP))
A. Reagents:
-----------
(1) PolydT35 1 ug/ul (Genset),
(2) Mouse COT-1 DNA (R) 1 mg/ml (Invitrogen, 500 ug, Cat. No. 18440-016),
(3) Human COT-1 DNA (R)1 mg/ml (Invitrogen, 500 ug, Cat. No. 15279-011),
(4) Microarray hybridization buffer (Amersham Pharmacia Biotech, Cat. No. RPK0325),
(5) Formamide (Sigma, Cat. No. F-9037). Use Mouse COT DNA on Mouse slides, Human COT DNA on Human slides and no COT on Arabidopsis slides.
B. Protocol for automatic hybridization with ASP:
------------------------------------------------
(1) Make 210 ul hybridization mixture per slide with 25% probe, 25% buffer and 50% formamide.
(2) Dissolve the Cy3 and Cy5 sample together in 12 ul of polydT35, 30 ul of Mouse or Human or no (Arabidopsis) COT, water to make a total volume of 52.5 ul, 52.5 ul of Microarray 4x hybridization buffer and 105 ul formamide.
(3) Denature at 96C for 3 minutes.
(4) Keep on ice until hybridization.
(5) Inject samples in ASP.
III. Scanning Protocol (VIB-MAF scanning v1)
A. Equipment:
------------
(1) Generation III Scanner, Amersham Biosciences
(2) Scanner software: Default scanner software
B. Scanning Protocol:
--------------------
(1) Before scanning, adjust scanning parameters to: Full Slide, Cy5 PMT=550V, Cy3 PMT=550V
Keywords = Thyroid
Keywords = Adenosine Receptor 2a
Lot batch = batch 36A, RPK 0331 ; Printed 30-Oct-2001

organism: Mus musculus

source_name: RDC-8 Thyroid knock-out (Cy5)

source_name: C57Bl6 Thyroid Wild Type (Cy3)


title: 3000002251

description:
I. Labelling Protocol (VIB-MAF Cy3 Labeling v1 and  VIB-MAF Cy5 Labeling v1)
[1] RNA amplification by in vitro transcription
This protocol has been published in L. G. Puskás, Á. Zvara, L. Hackler Jr., and P. Van Hummelen (2002). RNA amplification results in reproducible microarray data with slight ratio bias. Biotechniques (2002) vol. 32, no. 6 p.1330-1341.
1.1 Reagents:
------------
(1) RNase Out 40U/ul (Invitrogen, 5000U, Cat. No. 10777-019),
(2) HT7T primer, HPLC purified (Eurogentec, sequence: 5 - GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGGTTTTTTTTTT TTTTTTTTTTTTTT-3 ),
(3) Trehalose 1.7 M (Sigma, Cat. No. T-5251),
(4) 5 x first strand buffer (SuperScript II RT, Invitrogen, Cat. No. 18064-014),
(5) 0.1 M DTT (SuperScript II RT, Invitrogen, Cat. No. 18064-014),
(6) 10 mM dNTP-mix (Amersham Pharmacia Biotech, 100 mM, Cat. No. 27-2035- 01),
(7) SuperScript II RT 200U/ul (Invitrogen, 10000U, Cat. No. 18064-014),
(8) 5 x second strand buffer: 100 mM Tris-HCl (pH 6.9) (Invitrogen, Cat. No. 15714- 025 (Tris) and 1027 (HCl)), 450 mM KCl (Sigma, Cat. No. P-9541), 23 mM MgCl2 (Sigma, Cat. No. M 8266), 0.75 mM Beta-NAD+ (Sigma, Cat. No. N- 1636), 50 mM (NH4)2SO4 (Invitrogen, Cat. No. 15501-018)),
(9) E. coli DNA Ligase 10U/ul (Invitrogen, 100U, Cat. No. 18052-019),
(10) E. coli DNA Polymerase I 10U/ul (Invitrogen, 1000U, Cat. No. 18010-025),
(11) E. coli RNase H 2U/ul (Invitrogen, 120U, Cat. No. 18021-071),
(12) DEPC-water (Invitrogen, Cat. No. 10977-015),
(13) Ethanol Absolut (VWR, Cat. No. 1.08543.0250),
(14) QIAquick PCR Purification Kit (Qiagen, Cat. No 28106),
(15) NaAc 3M, pH 5.0 (Sigma, Cat. No. S-2889),
(16) AmpliScribeTM T7 Transcription Kit (Epicentre Technologies, Cat. No. AS3107),
(17) RNeasy Mini Kit (Qiagen, Cat. No 74106).
1.2 Protocol First Strand Synthesis:
-----------------------------------
(1) Mix 5 ug of total RNA (3 ul) with 1 ul of RNase Out 40U/ul.
(2) Add 1 ul of (HT7T) primer 200 ng/ul and 6 ul of 1.7 M Trehalose heated to 42C.
(3) Put on 75C for 5 minutes, chill on ice and spin down.
(4) Add 4 ul of 5 x first strand buffer, 2 ul of 0.1 M DTT, 1 ul of 10mM dNTP-mix, 1 ul of 1.7 M Trehalose and 1 ul of SuperScript II RT.
(5) Incubate at 37C for 5 minutes, at 45C for 5 minutes and then make 10 cycles of 60C for 2 minutes and 55C for 2 minutes. Go to 65C for 10 minutes and then go to 4C or chill on ice.
1.3 Protocol Second Strand Synthesis:
------------------------------------
(1) Add 33.4 ul of 5 x second strand buffer, 3.4 ul of 10 mM dNTP-mix, 1 ul of E. coli DNA Ligase (10U/ul), 4 ul of E. coli DNA Polymerase I (10U/ul), 1 ul of E. coli RNase H (2U/ul) and 103.8 ul of DEPC-water to the 20 ul of first strand mix.
(2) Incubate at 16C for 3 hours, at 65C for 10 minutes and then go to 4C or chill on ice.
(3) Purification with QIAquick PCR Purification Kit according to the manufacturers guidelines.
1.4 Protocol for In Vitro Transcription (IVT):
---------------------------------------------
(1) Add 2 ul of 10 x T7 reaction buffer (heated to 42C), 6 ul of 25 mM rNTP-mix, 2 ul of 100 mM DTT and 2 ul of AmpliScribe T7 enzyme solution to the DNA from second strand synthesis.
(2) Incubate at 37C for 3 hours, at 65C for 10 minutes and then go to 4C or chill on ice.
(3) Purification with QIAgen RNeasy Mini Kit according to the manufacturers guidelines.
(4) Check RNA concentration by OD. We recommend a 1/10 dilution in water or no dilution when using Nanodrop. Check A260/A280 ratio and determine the RNA concentration (1 OD = 40 ng/ul).
[2] Labeling
2.1 Reagents:
------------
(1) RNase Out 40U/ul (Invitrogen, 5000U, Cat. No. 10777-019),
(2) Random nonamers N9 1 ug/ul (Genset, 1862270),
(3) 5 x first strand buffer (SuperScript II RT, Invitrogen, Cat. No. 18064-014),
(4) 0.1 M DTT (SuperScript II RT, Invitrogen, Cat. No. 18064-014),
(5) 10 mM dNTP/2 mM dCTP (Amersham Pharmacia Biotech, 100 mM, Cat. No. 272050/60/70/80),
(6) CyTM-3-dCTP and CyTM-5-dCTP for microarrays (Amersham Pharmacia Biotech, 25 nmol, Cat. No. PA 53021 and PA 55021),
(7) SuperScript II RT 200U/ul (Invitrogen, 10000U, Cat. No. 18064-014),
(8) 2.5 M NaOH (Sigma, Cat. No. S-0899),
(9) MOPS (3-(N-Morpholino)propanesulfonic acid) (Sigma, Cat. No. M-8899),
(10) NaAc 3M, pH 5.0 (Sigma, Cat. No. S-2889),
(11) QIAquick PCR Purification Kit (Qiagen, Cat. No 28106).

2.2 Cy3 labeling Protocol:
----------------------
(1) Mix 5 ug of RNA from IVT (8 ul), 0.5 ul of RNase Out 40U/ul, 5 ul spikes (not for Arabidopsis thaliana) and 2 ul of random nonamers N9 1 ug/ul.
(2) Denaturate at 70C for 10 minutes, chill on ice and add the following components: 4 ul of first strand buffer, 1 ul of 10 mM dNTP/2 mM dCTP, 2 ul of 0.1 M DTT, 1.5 ul Cy3 dCTP and 1 ul of SuperScript II RT 200U/ul.
(3) Incubate at 42C for 2.5 hours and then go to 4C or chill on ice.
(4) Denaturate the RNA template by adding 2 ul of 2.5 M NaOH, incubating at 37C for exactly 15 minutes, adding 10 ul of 2 M MOPS and putting on ice.
(5) Purification with QIAquick PCR Purification Kit according to manufacturers guidelines.

2.3 Cy5 Labeling Protocol:
----------------------
(1) Mix 5 ug of RNA from IVT (8 ul), 0.5 ul of RNase Out 40U/ul, 5 ul spikes (not for Arabidopsis thaliana) and 2 ul of random nonamers N9 1 ug/ul.
(2) Denaturate at 70C for 10 minutes, chill on ice and add the following components: 4 ul of first strand buffer, 1 ul of 10 mM dNTP/2 mM dCTP, 2 ul of 0.1 M DTT, 1.5 ul of Cy5 dCTP and 1 ul of SuperScript II RT 200U/ul.
(3) Incubate at 42C for 2.5 hours and then go to 4C or chill on ice.
(4) Denaturate the RNA template by adding 2 ul of 2.5 M NaOH, incubating at 37C for exactly 15 minutes, adding 10 ul of 2 M MOPS and putting on ice.
(5) Purification with QIAquick PCR Purification Kit according to manufacturers guidelines.

II. Hybridisation Protocol (VIB-MAF Automatic hybridization v1 using Automatic Slide Processor (ASP))
A. Reagents:
-----------
(1) PolydT35 1 ug/ul (Genset),
(2) Mouse COT-1 DNA (R) 1 mg/ml (Invitrogen, 500 ug, Cat. No. 18440-016),
(3) Human COT-1 DNA (R)1 mg/ml (Invitrogen, 500 ug, Cat. No. 15279-011),
(4) Microarray hybridization buffer (Amersham Pharmacia Biotech, Cat. No. RPK0325),
(5) Formamide (Sigma, Cat. No. F-9037). Use Mouse COT DNA on Mouse slides, Human COT DNA on Human slides and no COT on Arabidopsis slides.
B. Protocol for automatic hybridization with ASP:
------------------------------------------------
(1) Make 210 ul hybridization mixture per slide with 25% probe, 25% buffer and 50% formamide.
(2) Dissolve the Cy3 and Cy5 sample together in 12 ul of polydT35, 30 ul of Mouse or Human or no (Arabidopsis) COT, water to make a total volume of 52.5 ul, 52.5 ul of Microarray 4x hybridization buffer and 105 ul formamide.
(3) Denature at 96C for 3 minutes.
(4) Keep on ice until hybridization.
(5) Inject samples in ASP.
III. Scanning Protocol (VIB-MAF scanning v1)
A. Equipment:
------------
(1) Generation III Scanner, Amersham Biosciences
(2) Scanner software: Default scanner software
B. Scanning Protocol:
--------------------
(1) Before scanning, adjust scanning parameters to: Full Slide, Cy5 PMT=550V, Cy3 PMT=550V
Keywords = Thyroid
Keywords = Adenosine Receptor 2a
Lot batch = batch 36, RPK 1331 ; Printed 1-Jun-2001


title: BLI_1_20857

status: Public on Nov 21 2003

city: Tsukuba City, Ibaraki

first: Chandana

last: Herath

address: Ikenodai 2

department: Development Biology

country: Japan

institute: National Institute of Agrobiological Sciences

description: Ten-microgram of pooled liver total RNA obtained from nonpregnant cycling cows was reverse transcribed in the presence of cyanine-5-fluorescent dye (Cy5)-conjugated dUTP (Amersham Pharmacia Biotech) by using Superscript II reverse transcriptase (Life Technologies, Rockville, MD) to yield Cy5 fluorescence-labeled control cDNA probe. Similarly, liver total RNA obtained on day 27 of a pregnant cow was separately reverse transcribed in the presence of Cy3-conjugated dUTP by using Superscript II reverse transcriptase to yield Cy3 fluorescence-labeled test cDNA probe. After a 2-h reverse transcription reaction at 42oC, RNA was degraded by addition of 1.5 µl of an alkaline solution (1N NaOH / 20 mM EDTA), and neutralized by addition of 1.5 µl 1N HCl. A labeled control cDNA probe and a test cDNA probe were then mixed and washed by centrifugation with TE (Tris-EDTA, pH 7.0) first and then with TE, 20 µg Salmon sperm DNA, 20 µg polyA and 20 µg yeast RNA in a Microcon YM-30 column (Millipore, Bedford, MA). The concentrated probe mix was adjusted with TE to a volume of 24.8 µl, denatured at 100oC for 2 min, spun for a few min and made to a final hybridization volume of 31 µl with 5.27 µl 20XSSC and 0.93 µl 10% SDS and kept at room temperature until used. Before hybridization, microarray glass slides were immersed in 0.2% SDS for 2 min followed by two washes each 2 min in autoclaved Milli Q water, and subjected to blocking in 180 ml of Methyl Pyrrolidinone (Aldrich) containing 2.7 g Succinic Anhydride (WAKO, Japan) and 15.3 ml 1M Boric acid (pH 8.0) solution for 20 min. Slides were then washed with autoclaved Milli Q water, and the spotted cDNA targets (microarray) were denatured in boiling water for 2 min. The slides were dehydrated in 100% ethanol for 2 min and air dried by spinning at a low speed. The mixture of labeled probes, for example, the control (Cy5) and test (Cy3), were then applied to the microarray, placed a cover slip carefully and incubated at 65oC for 14 – 16 h in a custom slide chamber with humidity maintained by a small reservoir of 3 x SSC. In addition, 3 - 6 µl of 3 x SSC was spotted at each corner of the slide, as far away from the array as possible. After incubation for 14 – 16 h at 65oC, the slides were washed in 2 x SSC / 0.03% SDS for 2 min, followed by washing in 2 x SSC, 1 x SSC and 0.2 x SSC, each for 2 min with gentle agitation, and air dried by spinning at a low speed.

organism: Bos taurus

source_name: Adult pregnant liver (day245-sample#2)

source_name: Adult nonpregnant liver


title: 3000054111

description:
I. Labelling Protocol (VIB-MAF Cy3 Labeling v1 and  VIB-MAF Cy5 Labeling v1)
[1] RNA amplification by in vitro transcription
This protocol has been published in L. G. Puskás, Á. Zvara, L. Hackler Jr., and P. Van Hummelen (2002). RNA amplification results in reproducible microarray data with slight ratio bias. Biotechniques (2002) vol. 32, no. 6 p.1330-1341.
1.1 Reagents:
------------
(1) RNase Out 40U/ul (Invitrogen, 5000U, Cat. No. 10777-019),
(2) HT7T primer, HPLC purified (Eurogentec, sequence: 5 - GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGGTTTTTTTTTT TTTTTTTTTTTTTT-3 ),
(3) Trehalose 1.7 M (Sigma, Cat. No. T-5251),
(4) 5 x first strand buffer (SuperScript II RT, Invitrogen, Cat. No. 18064-014),
(5) 0.1 M DTT (SuperScript II RT, Invitrogen, Cat. No. 18064-014),
(6) 10 mM dNTP-mix (Amersham Pharmacia Biotech, 100 mM, Cat. No. 27-2035- 01),
(7) SuperScript II RT 200U/ul (Invitrogen, 10000U, Cat. No. 18064-014),
(8) 5 x second strand buffer: 100 mM Tris-HCl (pH 6.9) (Invitrogen, Cat. No. 15714- 025 (Tris) and 1027 (HCl)), 450 mM KCl (Sigma, Cat. No. P-9541), 23 mM MgCl2 (Sigma, Cat. No. M 8266), 0.75 mM Beta-NAD+ (Sigma, Cat. No. N- 1636), 50 mM (NH4)2SO4 (Invitrogen, Cat. No. 15501-018)),
(9) E. coli DNA Ligase 10U/ul (Invitrogen, 100U, Cat. No. 18052-019),
(10) E. coli DNA Polymerase I 10U/ul (Invitrogen, 1000U, Cat. No. 18010-025),
(11) E. coli RNase H 2U/ul (Invitrogen, 120U, Cat. No. 18021-071),
(12) DEPC-water (Invitrogen, Cat. No. 10977-015),
(13) Ethanol Absolut (VWR, Cat. No. 1.08543.0250),
(14) QIAquick PCR Purification Kit (Qiagen, Cat. No 28106),
(15) NaAc 3M, pH 5.0 (Sigma, Cat. No. S-2889),
(16) AmpliScribeTM T7 Transcription Kit (Epicentre Technologies, Cat. No. AS3107),
(17) RNeasy Mini Kit (Qiagen, Cat. No 74106).
1.2 Protocol First Strand Synthesis:
-----------------------------------
(1) Mix 5 ug of total RNA (3 ul) with 1 ul of RNase Out 40U/ul.
(2) Add 1 ul of (HT7T) primer 200 ng/ul and 6 ul of 1.7 M Trehalose heated to 42C.
(3) Put on 75C for 5 minutes, chill on ice and spin down.
(4) Add 4 ul of 5 x first strand buffer, 2 ul of 0.1 M DTT, 1 ul of 10mM dNTP-mix, 1 ul of 1.7 M Trehalose and 1 ul of SuperScript II RT.
(5) Incubate at 37C for 5 minutes, at 45C for 5 minutes and then make 10 cycles of 60C for 2 minutes and 55C for 2 minutes. Go to 65C for 10 minutes and then go to 4C or chill on ice.
1.3 Protocol Second Strand Synthesis:
------------------------------------
(1) Add 33.4 ul of 5 x second strand buffer, 3.4 ul of 10 mM dNTP-mix, 1 ul of E. coli DNA Ligase (10U/ul), 4 ul of E. coli DNA Polymerase I (10U/ul), 1 ul of E. coli RNase H (2U/ul) and 103.8 ul of DEPC-water to the 20 ul of first strand mix.
(2) Incubate at 16C for 3 hours, at 65C for 10 minutes and then go to 4C or chill on ice.
(3) Purification with QIAquick PCR Purification Kit according to the manufacturers guidelines.
1.4 Protocol for In Vitro Transcription (IVT):
---------------------------------------------
(1) Add 2 ul of 10 x T7 reaction buffer (heated to 42C), 6 ul of 25 mM rNTP-mix, 2 ul of 100 mM DTT and 2 ul of AmpliScribe T7 enzyme solution to the DNA from second strand synthesis.
(2) Incubate at 37C for 3 hours, at 65C for 10 minutes and then go to 4C or chill on ice.
(3) Purification with QIAgen RNeasy Mini Kit according to the manufacturers guidelines.
(4) Check RNA concentration by OD. We recommend a 1/10 dilution in water or no dilution when using Nanodrop. Check A260/A280 ratio and determine the RNA concentration (1 OD = 40 ng/ul).
[2] Labeling
2.1 Reagents:
------------
(1) RNase Out 40U/ul (Invitrogen, 5000U, Cat. No. 10777-019),
(2) Random nonamers N9 1 ug/ul (Genset, 1862270),
(3) 5 x first strand buffer (SuperScript II RT, Invitrogen, Cat. No. 18064-014),
(4) 0.1 M DTT (SuperScript II RT, Invitrogen, Cat. No. 18064-014),
(5) 10 mM dNTP/2 mM dCTP (Amersham Pharmacia Biotech, 100 mM, Cat. No. 272050/60/70/80),
(6) CyTM-3-dCTP and CyTM-5-dCTP for microarrays (Amersham Pharmacia Biotech, 25 nmol, Cat. No. PA 53021 and PA 55021),
(7) SuperScript II RT 200U/ul (Invitrogen, 10000U, Cat. No. 18064-014),
(8) 2.5 M NaOH (Sigma, Cat. No. S-0899),
(9) MOPS (3-(N-Morpholino)propanesulfonic acid) (Sigma, Cat. No. M-8899),
(10) NaAc 3M, pH 5.0 (Sigma, Cat. No. S-2889),
(11) QIAquick PCR Purification Kit (Qiagen, Cat. No 28106).

2.2 Cy3 labeling Protocol:
----------------------
(1) Mix 5 ug of RNA from IVT (8 ul), 0.5 ul of RNase Out 40U/ul, 5 ul spikes (not for Arabidopsis thaliana) and 2 ul of random nonamers N9 1 ug/ul.
(2) Denaturate at 70C for 10 minutes, chill on ice and add the following components: 4 ul of first strand buffer, 1 ul of 10 mM dNTP/2 mM dCTP, 2 ul of 0.1 M DTT, 1.5 ul Cy3 dCTP and 1 ul of SuperScript II RT 200U/ul.
(3) Incubate at 42C for 2.5 hours and then go to 4C or chill on ice.
(4) Denaturate the RNA template by adding 2 ul of 2.5 M NaOH, incubating at 37C for exactly 15 minutes, adding 10 ul of 2 M MOPS and putting on ice.
(5) Purification with QIAquick PCR Purification Kit according to manufacturers guidelines.

2.3 Cy5 Labeling Protocol:
----------------------
(1) Mix 5 ug of RNA from IVT (8 ul), 0.5 ul of RNase Out 40U/ul, 5 ul spikes (not for Arabidopsis thaliana) and 2 ul of random nonamers N9 1 ug/ul.
(2) Denaturate at 70C for 10 minutes, chill on ice and add the following components: 4 ul of first strand buffer, 1 ul of 10 mM dNTP/2 mM dCTP, 2 ul of 0.1 M DTT, 1.5 ul of Cy5 dCTP and 1 ul of SuperScript II RT 200U/ul.
(3) Incubate at 42C for 2.5 hours and then go to 4C or chill on ice.
(4) Denaturate the RNA template by adding 2 ul of 2.5 M NaOH, incubating at 37C for exactly 15 minutes, adding 10 ul of 2 M MOPS and putting on ice.
(5) Purification with QIAquick PCR Purification Kit according to manufacturers guidelines.

II. Hybridisation Protocol (VIB-MAF Automatic hybridization v1 using Automatic Slide Processor (ASP))
A. Reagents:
-----------
(1) PolydT35 1 ug/ul (Genset),
(2) Mouse COT-1 DNA (R) 1 mg/ml (Invitrogen, 500 ug, Cat. No. 18440-016),
(3) Human COT-1 DNA (R)1 mg/ml (Invitrogen, 500 ug, Cat. No. 15279-011),
(4) Microarray hybridization buffer (Amersham Pharmacia Biotech, Cat. No. RPK0325),
(5) Formamide (Sigma, Cat. No. F-9037). Use Mouse COT DNA on Mouse slides, Human COT DNA on Human slides and no COT on Arabidopsis slides.
B. Protocol for automatic hybridization with ASP:
------------------------------------------------
(1) Make 210 ul hybridization mixture per slide with 25% probe, 25% buffer and 50% formamide.
(2) Dissolve the Cy3 and Cy5 sample together in 12 ul of polydT35, 30 ul of Mouse or Human or no (Arabidopsis) COT, water to make a total volume of 52.5 ul, 52.5 ul of Microarray 4x hybridization buffer and 105 ul formamide.
(3) Denature at 96C for 3 minutes.
(4) Keep on ice until hybridization.
(5) Inject samples in ASP.
III. Scanning Protocol (VIB-MAF scanning v1)
A. Equipment:
------------
(1) Generation III Scanner, Amersham Biosciences
(2) Scanner software: Default scanner software
B. Scanning Protocol:
--------------------
(1) Before scanning, adjust scanning parameters to: Full Slide, Cy5 PMT=550V, Cy3 PMT=550V
Keywords = Thyroid
Keywords = Adenosine Receptor 2a
Lot batch = type 7 Star Amersham ; lot 32 ; RPK 2331 ; Printed 23-Apr-2002

source_name: C57Bl6 Thyroid Wild Type (Cy5)

source_name: RDC-8 Thyroid knock-out (Cy3)


title: 24 hr A-chip 3-13-03


title: 3000002603

description:
I. Labelling Protocol (VIB-MAF Cy3 Labeling v1 and  VIB-MAF Cy5 Labeling v1)
[1] RNA amplification by in vitro transcription
This protocol has been published in L. G. Puskás, Á. Zvara, L. Hackler Jr., and P. Van Hummelen (2002). RNA amplification results in reproducible microarray data with slight ratio bias. Biotechniques (2002) vol. 32, no. 6 p.1330-1341.
1.1 Reagents:
------------
(1) RNase Out 40U/ul (Invitrogen, 5000U, Cat. No. 10777-019),
(2) HT7T primer, HPLC purified (Eurogentec, sequence: 5 - GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGGTTTTTTTTTT TTTTTTTTTTTTTT-3 ),
(3) Trehalose 1.7 M (Sigma, Cat. No. T-5251),
(4) 5 x first strand buffer (SuperScript II RT, Invitrogen, Cat. No. 18064-014),
(5) 0.1 M DTT (SuperScript II RT, Invitrogen, Cat. No. 18064-014),
(6) 10 mM dNTP-mix (Amersham Pharmacia Biotech, 100 mM, Cat. No. 27-2035- 01),
(7) SuperScript II RT 200U/ul (Invitrogen, 10000U, Cat. No. 18064-014),
(8) 5 x second strand buffer: 100 mM Tris-HCl (pH 6.9) (Invitrogen, Cat. No. 15714- 025 (Tris) and 1027 (HCl)), 450 mM KCl (Sigma, Cat. No. P-9541), 23 mM MgCl2 (Sigma, Cat. No. M 8266), 0.75 mM Beta-NAD+ (Sigma, Cat. No. N- 1636), 50 mM (NH4)2SO4 (Invitrogen, Cat. No. 15501-018)),
(9) E. coli DNA Ligase 10U/ul (Invitrogen, 100U, Cat. No. 18052-019),
(10) E. coli DNA Polymerase I 10U/ul (Invitrogen, 1000U, Cat. No. 18010-025),
(11) E. coli RNase H 2U/ul (Invitrogen, 120U, Cat. No. 18021-071),
(12) DEPC-water (Invitrogen, Cat. No. 10977-015),
(13) Ethanol Absolut (VWR, Cat. No. 1.08543.0250),
(14) QIAquick PCR Purification Kit (Qiagen, Cat. No 28106),
(15) NaAc 3M, pH 5.0 (Sigma, Cat. No. S-2889),
(16) AmpliScribeTM T7 Transcription Kit (Epicentre Technologies, Cat. No. AS3107),
(17) RNeasy Mini Kit (Qiagen, Cat. No 74106).
1.2 Protocol First Strand Synthesis:
-----------------------------------
(1) Mix 5 ug of total RNA (3 ul) with 1 ul of RNase Out 40U/ul.
(2) Add 1 ul of (HT7T) primer 200 ng/ul and 6 ul of 1.7 M Trehalose heated to 42C.
(3) Put on 75C for 5 minutes, chill on ice and spin down.
(4) Add 4 ul of 5 x first strand buffer, 2 ul of 0.1 M DTT, 1 ul of 10mM dNTP-mix, 1 ul of 1.7 M Trehalose and 1 ul of SuperScript II RT.
(5) Incubate at 37C for 5 minutes, at 45C for 5 minutes and then make 10 cycles of 60C for 2 minutes and 55C for 2 minutes. Go to 65C for 10 minutes and then go to 4C or chill on ice.
1.3 Protocol Second Strand Synthesis:
------------------------------------
(1) Add 33.4 ul of 5 x second strand buffer, 3.4 ul of 10 mM dNTP-mix, 1 ul of E. coli DNA Ligase (10U/ul), 4 ul of E. coli DNA Polymerase I (10U/ul), 1 ul of E. coli RNase H (2U/ul) and 103.8 ul of DEPC-water to the 20 ul of first strand mix.
(2) Incubate at 16C for 3 hours, at 65C for 10 minutes and then go to 4C or chill on ice.
(3) Purification with QIAquick PCR Purification Kit according to the manufacturers guidelines.
1.4 Protocol for In Vitro Transcription (IVT):
---------------------------------------------
(1) Add 2 ul of 10 x T7 reaction buffer (heated to 42C), 6 ul of 25 mM rNTP-mix, 2 ul of 100 mM DTT and 2 ul of AmpliScribe T7 enzyme solution to the DNA from second strand synthesis.
(2) Incubate at 37C for 3 hours, at 65C for 10 minutes and then go to 4C or chill on ice.
(3) Purification with QIAgen RNeasy Mini Kit according to the manufacturers guidelines.
(4) Check RNA concentration by OD. We recommend a 1/10 dilution in water or no dilution when using Nanodrop. Check A260/A280 ratio and determine the RNA concentration (1 OD = 40 ng/ul).
[2] Labeling
2.1 Reagents:
------------
(1) RNase Out 40U/ul (Invitrogen, 5000U, Cat. No. 10777-019),
(2) Random nonamers N9 1 ug/ul (Genset, 1862270),
(3) 5 x first strand buffer (SuperScript II RT, Invitrogen, Cat. No. 18064-014),
(4) 0.1 M DTT (SuperScript II RT, Invitrogen, Cat. No. 18064-014),
(5) 10 mM dNTP/2 mM dCTP (Amersham Pharmacia Biotech, 100 mM, Cat. No. 272050/60/70/80),
(6) CyTM-3-dCTP and CyTM-5-dCTP for microarrays (Amersham Pharmacia Biotech, 25 nmol, Cat. No. PA 53021 and PA 55021),
(7) SuperScript II RT 200U/ul (Invitrogen, 10000U, Cat. No. 18064-014),
(8) 2.5 M NaOH (Sigma, Cat. No. S-0899),
(9) MOPS (3-(N-Morpholino)propanesulfonic acid) (Sigma, Cat. No. M-8899),
(10) NaAc 3M, pH 5.0 (Sigma, Cat. No. S-2889),
(11) QIAquick PCR Purification Kit (Qiagen, Cat. No 28106).

2.2 Cy3 labeling Protocol:
----------------------
(1) Mix 5 ug of RNA from IVT (8 ul), 0.5 ul of RNase Out 40U/ul, 5 ul spikes (not for Arabidopsis thaliana) and 2 ul of random nonamers N9 1 ug/ul.
(2) Denaturate at 70C for 10 minutes, chill on ice and add the following components: 4 ul of first strand buffer, 1 ul of 10 mM dNTP/2 mM dCTP, 2 ul of 0.1 M DTT, 1.5 ul Cy3 dCTP and 1 ul of SuperScript II RT 200U/ul.
(3) Incubate at 42C for 2.5 hours and then go to 4C or chill on ice.
(4) Denaturate the RNA template by adding 2 ul of 2.5 M NaOH, incubating at 37C for exactly 15 minutes, adding 10 ul of 2 M MOPS and putting on ice.
(5) Purification with QIAquick PCR Purification Kit according to manufacturers guidelines.

2.3 Cy5 Labeling Protocol:
----------------------
(1) Mix 5 ug of RNA from IVT (8 ul), 0.5 ul of RNase Out 40U/ul, 5 ul spikes (not for Arabidopsis thaliana) and 2 ul of random nonamers N9 1 ug/ul.
(2) Denaturate at 70C for 10 minutes, chill on ice and add the following components: 4 ul of first strand buffer, 1 ul of 10 mM dNTP/2 mM dCTP, 2 ul of 0.1 M DTT, 1.5 ul of Cy5 dCTP and 1 ul of SuperScript II RT 200U/ul.
(3) Incubate at 42C for 2.5 hours and then go to 4C or chill on ice.
(4) Denaturate the RNA template by adding 2 ul of 2.5 M NaOH, incubating at 37C for exactly 15 minutes, adding 10 ul of 2 M MOPS and putting on ice.
(5) Purification with QIAquick PCR Purification Kit according to manufacturers guidelines.

II. Hybridisation Protocol (VIB-MAF Automatic hybridization v1 using Automatic Slide Processor (ASP))
A. Reagents:
-----------
(1) PolydT35 1 ug/ul (Genset),
(2) Mouse COT-1 DNA (R) 1 mg/ml (Invitrogen, 500 ug, Cat. No. 18440-016),
(3) Human COT-1 DNA (R)1 mg/ml (Invitrogen, 500 ug, Cat. No. 15279-011),
(4) Microarray hybridization buffer (Amersham Pharmacia Biotech, Cat. No. RPK0325),
(5) Formamide (Sigma, Cat. No. F-9037). Use Mouse COT DNA on Mouse slides, Human COT DNA on Human slides and no COT on Arabidopsis slides.
B. Protocol for automatic hybridization with ASP:
------------------------------------------------
(1) Make 210 ul hybridization mixture per slide with 25% probe, 25% buffer and 50% formamide.
(2) Dissolve the Cy3 and Cy5 sample together in 12 ul of polydT35, 30 ul of Mouse or Human or no (Arabidopsis) COT, water to make a total volume of 52.5 ul, 52.5 ul of Microarray 4x hybridization buffer and 105 ul formamide.
(3) Denature at 96C for 3 minutes.
(4) Keep on ice until hybridization.
(5) Inject samples in ASP.
III. Scanning Protocol (VIB-MAF scanning v1)
A. Equipment:
------------
(1) Generation III Scanner, Amersham Biosciences
(2) Scanner software: Default scanner software
B. Scanning Protocol:
--------------------
(1) Before scanning, adjust scanning parameters to: Full Slide, Cy5 PMT=550V, Cy3 PMT=550V
Keywords = Thyroid
Keywords = Adenosine Receptor 2a
Lot batch = batch 36A, RPK 0331 ; Printed 24-Sep-2001


title: 12 hr B-chip 6-11-02

description: IB3-1 (deltaF508/W1282X) bronchial epithelial cells were cultured in T75 flasks with gentamicin-free LHC-8 medium.  After reaching 80% confluence cells were treated with 1 mM PBA for 12 hr.  A T75 flask of confluent IB3-1 cells was rinsed twice with ice cold Hank’s buffer then scraped into 3ml of ice cold TRIzol (Gibco BRL) then rinsed with 3 ml ice cold TRIzol and the mRNA was isolated according to the TRIzol protocol.  Array images were assessed by eye to confirm scanner alignment and the absence of significant bubbles or scratches.  Chip scanning and expression analysis was performed using Microarray Analysis Suite 5.0 software and scaled to an average probe set intensity of 150.


title: BDL_3hr_2

status: Public on Dec 13 2003

city: Chiba

first: Kensuke

last: Kojima

state: Chiba

department: Graduate School of Pharmaceutical Sciences

institute: Chiba University

description:
Male Sprague-Dawley rats weighing 250-300 g were purchased from Japan SLC Co. (Shizuoka, Japan). The animals were housed under a daily controlled 12-h light and 12-h dark cycle at 23 °C with free access to rat chow (Japan SLC Co.) and water for 1 week prior to the experiments.
Rats were laparotomized under ether anesthesia in each experiment, and the common bile duct of each rat was ligated between the liver hilus and small intestine. Control animals underwent a sham operation with exposure but without ligation of the common bile duct. After the CBDL (common bile duct ligation) or sham operation, animals were allowed access to food and water ad libitum.
Groups of three rats were sacrificed under ether anesthesia 3 hr after the CBDL or sham operation. At the time of sacrifice, the right lateral liver lobe was removed and flash-frozen in liquid nitrogen and stored at -80 °C. Total RNA isolated from frozen livers using TRIzol reagent (Invitrogen Co., Carlsbad, CA) was mixed to minimize variation among animals. Poly(A) RNA was purified using Oligotex-dT30 (Takara Shuzo Co., Ltd., Kyoto, Japan) in accordance with manufacturer’s instructions. Fluorescence-labeled probes were prepared by reverse transcription using a superscript II reverse transcriptase (Invitrogen Co.) and cyanine3- and cyanine5-dUTP (Perkin-Elmer Inc., Wellesley, MA). Poly(A) RNAs derived from rats that has undergone CBDL and from those that has undergone a sham operation were labeled with cyanine-3 and cyanine-5, respectively. Fluorescence-labeled probes were purified using a MinElute PCR Products Purification Kit (Quiagen GmbH, Hilden, Germany) in accordance with manufacturer’s instructions. Each purified probe was suspended in hybridization buffer containing 1.6 mg/mL poly(A) (Roche Diagnostics, Basel, Switzerland) and yeast tRNA (Roche Diagnostics), 0.67 mg/mL herring sperm, 16% 20 x SSC and 0.3% SDS and applied to a cDNA microarray containing 1,800 rat genes on a slide glass (Asahi Technoglass Co., Tokyo, Japan). Three house-keeping genes (GAPDH, HPRT and b-actin) and rat unrelated traits (Lambda DNA) were also spotted on the slide as internal positive and negative controls, respectively.
The microarray was cover-slipped and incubated in a sealed chamber (Asahi Technoglass Co.) for 16 hrs under a humidified condition. After being washed in low-stringency buffer (2 x SSC and 0.1% SDS), high-stringency buffer (0.2 x SSC and 0.1% SDS) and 0.2 x SSC and then rinsing with 99.5% ethanol, the slide was dried by centrifugation at low speed and used for scanning. Scanning and quantification. Fluorescence of cyanine-3 and cyanine-5 was scanned by using a ScanArray 4000 (Packard BioChip Technologies, Billerica, MA) and quantified by using QuantArray Software (Packard BioChip Technologies, Billerica, MA). Correction of spot intensity was made by subtraction of the background, and was normalized by the average of the intensity of GAPDH. The ratios of intensities in the samples delived from CBDL rats and these in the samples derived from sham operation rats were calculated for each transcript and log2-transformed.

organism: Rattus norvegicus

source_name: Sham operation_Cy5_3hr

source_name: Common bile duct ligation_Cy3_3hr


title: 24 hr B-chip 1-21-03


title: 12 hr B-chip 3-13-03


title: 24 hr A-chip 1-21-03


title: 24 hr A-chip 6-13-02


title: 3000002968

description:
I. Labelling Protocol (VIB-MAF Cy3 Labeling v1 and  VIB-MAF Cy5 Labeling v1)
[1] RNA amplification by in vitro transcription
This protocol has been published in L. G. Puskás, Á. Zvara, L. Hackler Jr., and P. Van Hummelen (2002). RNA amplification results in reproducible microarray data with slight ratio bias. Biotechniques (2002) vol. 32, no. 6 p.1330-1341.
1.1 Reagents:
------------
(1) RNase Out 40U/ul (Invitrogen, 5000U, Cat. No. 10777-019),
(2) HT7T primer, HPLC purified (Eurogentec, sequence: 5 - GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGGTTTTTTTTTT TTTTTTTTTTTTTT-3 ),
(3) Trehalose 1.7 M (Sigma, Cat. No. T-5251),
(4) 5 x first strand buffer (SuperScript II RT, Invitrogen, Cat. No. 18064-014),
(5) 0.1 M DTT (SuperScript II RT, Invitrogen, Cat. No. 18064-014),
(6) 10 mM dNTP-mix (Amersham Pharmacia Biotech, 100 mM, Cat. No. 27-2035- 01),
(7) SuperScript II RT 200U/ul (Invitrogen, 10000U, Cat. No. 18064-014),
(8) 5 x second strand buffer: 100 mM Tris-HCl (pH 6.9) (Invitrogen, Cat. No. 15714- 025 (Tris) and 1027 (HCl)), 450 mM KCl (Sigma, Cat. No. P-9541), 23 mM MgCl2 (Sigma, Cat. No. M 8266), 0.75 mM Beta-NAD+ (Sigma, Cat. No. N- 1636), 50 mM (NH4)2SO4 (Invitrogen, Cat. No. 15501-018)),
(9) E. coli DNA Ligase 10U/ul (Invitrogen, 100U, Cat. No. 18052-019),
(10) E. coli DNA Polymerase I 10U/ul (Invitrogen, 1000U, Cat. No. 18010-025),
(11) E. coli RNase H 2U/ul (Invitrogen, 120U, Cat. No. 18021-071),
(12) DEPC-water (Invitrogen, Cat. No. 10977-015),
(13) Ethanol Absolut (VWR, Cat. No. 1.08543.0250),
(14) QIAquick PCR Purification Kit (Qiagen, Cat. No 28106),
(15) NaAc 3M, pH 5.0 (Sigma, Cat. No. S-2889),
(16) AmpliScribeTM T7 Transcription Kit (Epicentre Technologies, Cat. No. AS3107),
(17) RNeasy Mini Kit (Qiagen, Cat. No 74106).
1.2 Protocol First Strand Synthesis:
-----------------------------------
(1) Mix 5 ug of total RNA (3 ul) with 1 ul of RNase Out 40U/ul.
(2) Add 1 ul of (HT7T) primer 200 ng/ul and 6 ul of 1.7 M Trehalose heated to 42C.
(3) Put on 75C for 5 minutes, chill on ice and spin down.
(4) Add 4 ul of 5 x first strand buffer, 2 ul of 0.1 M DTT, 1 ul of 10mM dNTP-mix, 1 ul of 1.7 M Trehalose and 1 ul of SuperScript II RT.
(5) Incubate at 37C for 5 minutes, at 45C for 5 minutes and then make 10 cycles of 60C for 2 minutes and 55C for 2 minutes. Go to 65C for 10 minutes and then go to 4C or chill on ice.
1.3 Protocol Second Strand Synthesis:
------------------------------------
(1) Add 33.4 ul of 5 x second strand buffer, 3.4 ul of 10 mM dNTP-mix, 1 ul of E. coli DNA Ligase (10U/ul), 4 ul of E. coli DNA Polymerase I (10U/ul), 1 ul of E. coli RNase H (2U/ul) and 103.8 ul of DEPC-water to the 20 ul of first strand mix.
(2) Incubate at 16C for 3 hours, at 65C for 10 minutes and then go to 4C or chill on ice.
(3) Purification with QIAquick PCR Purification Kit according to the manufacturers guidelines.
1.4 Protocol for In Vitro Transcription (IVT):
---------------------------------------------
(1) Add 2 ul of 10 x T7 reaction buffer (heated to 42C), 6 ul of 25 mM rNTP-mix, 2 ul of 100 mM DTT and 2 ul of AmpliScribe T7 enzyme solution to the DNA from second strand synthesis.
(2) Incubate at 37C for 3 hours, at 65C for 10 minutes and then go to 4C or chill on ice.
(3) Purification with QIAgen RNeasy Mini Kit according to the manufacturers guidelines.
(4) Check RNA concentration by OD. We recommend a 1/10 dilution in water or no dilution when using Nanodrop. Check A260/A280 ratio and determine the RNA concentration (1 OD = 40 ng/ul).
[2] Labeling
2.1 Reagents:
------------
(1) RNase Out 40U/ul (Invitrogen, 5000U, Cat. No. 10777-019),
(2) Random nonamers N9 1 ug/ul (Genset, 1862270),
(3) 5 x first strand buffer (SuperScript II RT, Invitrogen, Cat. No. 18064-014),
(4) 0.1 M DTT (SuperScript II RT, Invitrogen, Cat. No. 18064-014),
(5) 10 mM dNTP/2 mM dCTP (Amersham Pharmacia Biotech, 100 mM, Cat. No. 272050/60/70/80),
(6) CyTM-3-dCTP and CyTM-5-dCTP for microarrays (Amersham Pharmacia Biotech, 25 nmol, Cat. No. PA 53021 and PA 55021),
(7) SuperScript II RT 200U/ul (Invitrogen, 10000U, Cat. No. 18064-014),
(8) 2.5 M NaOH (Sigma, Cat. No. S-0899),
(9) MOPS (3-(N-Morpholino)propanesulfonic acid) (Sigma, Cat. No. M-8899),
(10) NaAc 3M, pH 5.0 (Sigma, Cat. No. S-2889),
(11) QIAquick PCR Purification Kit (Qiagen, Cat. No 28106).

2.2 Cy3 labeling Protocol:
----------------------
(1) Mix 5 ug of RNA from IVT (8 ul), 0.5 ul of RNase Out 40U/ul, 5 ul spikes (not for Arabidopsis thaliana) and 2 ul of random nonamers N9 1 ug/ul.
(2) Denaturate at 70C for 10 minutes, chill on ice and add the following components: 4 ul of first strand buffer, 1 ul of 10 mM dNTP/2 mM dCTP, 2 ul of 0.1 M DTT, 1.5 ul Cy3 dCTP and 1 ul of SuperScript II RT 200U/ul.
(3) Incubate at 42C for 2.5 hours and then go to 4C or chill on ice.
(4) Denaturate the RNA template by adding 2 ul of 2.5 M NaOH, incubating at 37C for exactly 15 minutes, adding 10 ul of 2 M MOPS and putting on ice.
(5) Purification with QIAquick PCR Purification Kit according to manufacturers guidelines.

2.3 Cy5 Labeling Protocol:
----------------------
(1) Mix 5 ug of RNA from IVT (8 ul), 0.5 ul of RNase Out 40U/ul, 5 ul spikes (not for Arabidopsis thaliana) and 2 ul of random nonamers N9 1 ug/ul.
(2) Denaturate at 70C for 10 minutes, chill on ice and add the following components: 4 ul of first strand buffer, 1 ul of 10 mM dNTP/2 mM dCTP, 2 ul of 0.1 M DTT, 1.5 ul of Cy5 dCTP and 1 ul of SuperScript II RT 200U/ul.
(3) Incubate at 42C for 2.5 hours and then go to 4C or chill on ice.
(4) Denaturate the RNA template by adding 2 ul of 2.5 M NaOH, incubating at 37C for exactly 15 minutes, adding 10 ul of 2 M MOPS and putting on ice.
(5) Purification with QIAquick PCR Purification Kit according to manufacturers guidelines.

II. Hybridisation Protocol (VIB-MAF Automatic hybridization v1 using Automatic Slide Processor (ASP))
A. Reagents:
-----------
(1) PolydT35 1 ug/ul (Genset),
(2) Mouse COT-1 DNA (R) 1 mg/ml (Invitrogen, 500 ug, Cat. No. 18440-016),
(3) Human COT-1 DNA (R)1 mg/ml (Invitrogen, 500 ug, Cat. No. 15279-011),
(4) Microarray hybridization buffer (Amersham Pharmacia Biotech, Cat. No. RPK0325),
(5) Formamide (Sigma, Cat. No. F-9037). Use Mouse COT DNA on Mouse slides, Human COT DNA on Human slides and no COT on Arabidopsis slides.
B. Protocol for automatic hybridization with ASP:
------------------------------------------------
(1) Make 210 ul hybridization mixture per slide with 25% probe, 25% buffer and 50% formamide.
(2) Dissolve the Cy3 and Cy5 sample together in 12 ul of polydT35, 30 ul of Mouse or Human or no (Arabidopsis) COT, water to make a total volume of 52.5 ul, 52.5 ul of Microarray 4x hybridization buffer and 105 ul formamide.
(3) Denature at 96C for 3 minutes.
(4) Keep on ice until hybridization.
(5) Inject samples in ASP.
III. Scanning Protocol (VIB-MAF scanning v1)
A. Equipment:
------------
(1) Generation III Scanner, Amersham Biosciences
(2) Scanner software: Default scanner software
B. Scanning Protocol:
--------------------
(1) Before scanning, adjust scanning parameters to: Full Slide, Cy5 PMT=550V, Cy3 PMT=550V
Keywords = Thyroid
Keywords = Adenosine Receptor 2a
Lot batch = batch 36A, RPK 0331 ; Printed 9-Oct-2001


title: BLI_1_20867

source_name: Adult pregnant liver (day28)


title: 12 hr A-chip 3-13-03


title: 12 hr B-chip 1-21-03


title: 6000028045

description:
I. Labelling Protocol (VIB-MAF Cy3 Labeling v1 and  VIB-MAF Cy5 Labeling v1)
[1] RNA amplification by in vitro transcription
This protocol has been published in L. G. Puskás, Á. Zvara, L. Hackler Jr., and P. Van Hummelen (2002). RNA amplification results in reproducible microarray data with slight ratio bias. Biotechniques (2002) vol. 32, no. 6 p.1330-1341.
1.1 Reagents:
------------
(1) RNase Out 40U/ul (Invitrogen, 5000U, Cat. No. 10777-019),
(2) HT7T primer, HPLC purified (Eurogentec, sequence: 5 - GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGGTTTTTTTTTT TTTTTTTTTTTTTT-3 ),
(3) Trehalose 1.7 M (Sigma, Cat. No. T-5251),
(4) 5 x first strand buffer (SuperScript II RT, Invitrogen, Cat. No. 18064-014),
(5) 0.1 M DTT (SuperScript II RT, Invitrogen, Cat. No. 18064-014),
(6) 10 mM dNTP-mix (Amersham Pharmacia Biotech, 100 mM, Cat. No. 27-2035- 01),
(7) SuperScript II RT 200U/ul (Invitrogen, 10000U, Cat. No. 18064-014),
(8) 5 x second strand buffer: 100 mM Tris-HCl (pH 6.9) (Invitrogen, Cat. No. 15714- 025 (Tris) and 1027 (HCl)), 450 mM KCl (Sigma, Cat. No. P-9541), 23 mM MgCl2 (Sigma, Cat. No. M 8266), 0.75 mM Beta-NAD+ (Sigma, Cat. No. N- 1636), 50 mM (NH4)2SO4 (Invitrogen, Cat. No. 15501-018)),
(9) E. coli DNA Ligase 10U/ul (Invitrogen, 100U, Cat. No. 18052-019),
(10) E. coli DNA Polymerase I 10U/ul (Invitrogen, 1000U, Cat. No. 18010-025),
(11) E. coli RNase H 2U/ul (Invitrogen, 120U, Cat. No. 18021-071),
(12) DEPC-water (Invitrogen, Cat. No. 10977-015),
(13) Ethanol Absolut (VWR, Cat. No. 1.08543.0250),
(14) QIAquick PCR Purification Kit (Qiagen, Cat. No 28106),
(15) NaAc 3M, pH 5.0 (Sigma, Cat. No. S-2889),
(16) AmpliScribeTM T7 Transcription Kit (Epicentre Technologies, Cat. No. AS3107),
(17) RNeasy Mini Kit (Qiagen, Cat. No 74106).
1.2 Protocol First Strand Synthesis:
-----------------------------------
(1) Mix 5 ug of total RNA (3 ul) with 1 ul of RNase Out 40U/ul.
(2) Add 1 ul of (HT7T) primer 200 ng/ul and 6 ul of 1.7 M Trehalose heated to 42C.
(3) Put on 75C for 5 minutes, chill on ice and spin down.
(4) Add 4 ul of 5 x first strand buffer, 2 ul of 0.1 M DTT, 1 ul of 10mM dNTP-mix, 1 ul of 1.7 M Trehalose and 1 ul of SuperScript II RT.
(5) Incubate at 37C for 5 minutes, at 45C for 5 minutes and then make 10 cycles of 60C for 2 minutes and 55C for 2 minutes. Go to 65C for 10 minutes and then go to 4C or chill on ice.
1.3 Protocol Second Strand Synthesis:
------------------------------------
(1) Add 33.4 ul of 5 x second strand buffer, 3.4 ul of 10 mM dNTP-mix, 1 ul of E. coli DNA Ligase (10U/ul), 4 ul of E. coli DNA Polymerase I (10U/ul), 1 ul of E. coli RNase H (2U/ul) and 103.8 ul of DEPC-water to the 20 ul of first strand mix.
(2) Incubate at 16C for 3 hours, at 65C for 10 minutes and then go to 4C or chill on ice.
(3) Purification with QIAquick PCR Purification Kit according to the manufacturers guidelines.
1.4 Protocol for In Vitro Transcription (IVT):
---------------------------------------------
(1) Add 2 ul of 10 x T7 reaction buffer (heated to 42C), 6 ul of 25 mM rNTP-mix, 2 ul of 100 mM DTT and 2 ul of AmpliScribe T7 enzyme solution to the DNA from second strand synthesis.
(2) Incubate at 37C for 3 hours, at 65C for 10 minutes and then go to 4C or chill on ice.
(3) Purification with QIAgen RNeasy Mini Kit according to the manufacturers guidelines.
(4) Check RNA concentration by OD. We recommend a 1/10 dilution in water or no dilution when using Nanodrop. Check A260/A280 ratio and determine the RNA concentration (1 OD = 40 ng/ul).
[2] Labeling
2.1 Reagents:
------------
(1) RNase Out 40U/ul (Invitrogen, 5000U, Cat. No. 10777-019),
(2) Random nonamers N9 1 ug/ul (Genset, 1862270),
(3) 5 x first strand buffer (SuperScript II RT, Invitrogen, Cat. No. 18064-014),
(4) 0.1 M DTT (SuperScript II RT, Invitrogen, Cat. No. 18064-014),
(5) 10 mM dNTP/2 mM dCTP (Amersham Pharmacia Biotech, 100 mM, Cat. No. 272050/60/70/80),
(6) CyTM-3-dCTP and CyTM-5-dCTP for microarrays (Amersham Pharmacia Biotech, 25 nmol, Cat. No. PA 53021 and PA 55021),
(7) SuperScript II RT 200U/ul (Invitrogen, 10000U, Cat. No. 18064-014),
(8) 2.5 M NaOH (Sigma, Cat. No. S-0899),
(9) MOPS (3-(N-Morpholino)propanesulfonic acid) (Sigma, Cat. No. M-8899),
(10) NaAc 3M, pH 5.0 (Sigma, Cat. No. S-2889),
(11) QIAquick PCR Purification Kit (Qiagen, Cat. No 28106).

2.2 Cy3 labeling Protocol:
----------------------
(1) Mix 5 ug of RNA from IVT (8 ul), 0.5 ul of RNase Out 40U/ul, 5 ul spikes (not for Arabidopsis thaliana) and 2 ul of random nonamers N9 1 ug/ul.
(2) Denaturate at 70C for 10 minutes, chill on ice and add the following components: 4 ul of first strand buffer, 1 ul of 10 mM dNTP/2 mM dCTP, 2 ul of 0.1 M DTT, 1.5 ul Cy3 dCTP and 1 ul of SuperScript II RT 200U/ul.
(3) Incubate at 42C for 2.5 hours and then go to 4C or chill on ice.
(4) Denaturate the RNA template by adding 2 ul of 2.5 M NaOH, incubating at 37C for exactly 15 minutes, adding 10 ul of 2 M MOPS and putting on ice.
(5) Purification with QIAquick PCR Purification Kit according to manufacturers guidelines.

2.3 Cy5 Labeling Protocol:
----------------------
(1) Mix 5 ug of RNA from IVT (8 ul), 0.5 ul of RNase Out 40U/ul, 5 ul spikes (not for Arabidopsis thaliana) and 2 ul of random nonamers N9 1 ug/ul.
(2) Denaturate at 70C for 10 minutes, chill on ice and add the following components: 4 ul of first strand buffer, 1 ul of 10 mM dNTP/2 mM dCTP, 2 ul of 0.1 M DTT, 1.5 ul of Cy5 dCTP and 1 ul of SuperScript II RT 200U/ul.
(3) Incubate at 42C for 2.5 hours and then go to 4C or chill on ice.
(4) Denaturate the RNA template by adding 2 ul of 2.5 M NaOH, incubating at 37C for exactly 15 minutes, adding 10 ul of 2 M MOPS and putting on ice.
(5) Purification with QIAquick PCR Purification Kit according to manufacturers guidelines.

II. Hybridisation Protocol (VIB-MAF Automatic hybridization v1 using Automatic Slide Processor (ASP))
A. Reagents:
-----------
(1) PolydT35 1 ug/ul (Genset),
(2) Mouse COT-1 DNA (R) 1 mg/ml (Invitrogen, 500 ug, Cat. No. 18440-016),
(3) Human COT-1 DNA (R)1 mg/ml (Invitrogen, 500 ug, Cat. No. 15279-011),
(4) Microarray hybridization buffer (Amersham Pharmacia Biotech, Cat. No. RPK0325),
(5) Formamide (Sigma, Cat. No. F-9037). Use Mouse COT DNA on Mouse slides, Human COT DNA on Human slides and no COT on Arabidopsis slides.
B. Protocol for automatic hybridization with ASP:
------------------------------------------------
(1) Make 210 ul hybridization mixture per slide with 25% probe, 25% buffer and 50% formamide.
(2) Dissolve the Cy3 and Cy5 sample together in 12 ul of polydT35, 30 ul of Mouse or Human or no (Arabidopsis) COT, water to make a total volume of 52.5 ul, 52.5 ul of Microarray 4x hybridization buffer and 105 ul formamide.
(3) Denature at 96C for 3 minutes.
(4) Keep on ice until hybridization.
(5) Inject samples in ASP.
III. Scanning Protocol (VIB-MAF scanning v1)
A. Equipment:
------------
(1) Generation III Scanner, Amersham Biosciences
(2) Scanner software: Default scanner software
B. Scanning Protocol:
--------------------
(1) Before scanning, adjust scanning parameters to: Full Slide, Cy5 PMT=550V, Cy3 PMT=550V
Keywords = Thyroid
Keywords = Adenosine Receptor 2a
Lot batch = Printed 3-May-2001


title: 12 hr A-chip 1-21-03


title: Endophyte infected, rep 3

status: Public on Oct 26 2003

city: Carbondale

first: M.

middle: Javed

last: Iqbal

state: IL

address: 176 Ag. Building, MC 4415

department: Animal Sci, Food & Nutrition + Plant, Soil & General Agriculture

institute: Southern Illinois University at Carbondale

description:
Gene expression analysis in heifer luteal tissue by interspecies microarray analysis.
Post-pubertal heifer fed diet containing endophyte infected fescue seed. Heifer was slaughtered at a local abattoir 14 days after ovulation and used for this study. Ovaries were collected and shipped at room temperature to the laboratory.  Approximately 3 mm3 samples of luteal tissue were placed in cryovials and frozen at -80oC until processed.
One sample was ground to a fine powder in liquid N and total RNA was extracted using the RNeasy® Mini Kit (Qiagen, Valencia, CA).  Quality and quantity of RNA was determined by gel electrophoresis and spectrophotometry.
Five µg of total RNA was converted to cDNA and then biotin-labeled cRNA by linear amplification (CodeLink® Expression Bioarrays, Amersham Biosciences Corp, Piscataway, NJ, USA). Ten micrograms of labeled cRNA were hybridized to CodeLink UniSet Rat I Bioarray (Amersham Biosciences Corp, Piscataway, NJ, USA) by the Amersham Biosciences Facility.

source_name: Ovary (luteal tissue); EI3-T00168440


title: Endophyte infected + domperidone, rep 3

description:
Gene expression analysis in heifer luteal tissue by interspecies microarray analysis.
Post-pubertal heifer fed diet containing endophyte infected fescue seed supplemented with 1.44 mg domperidone/kg body weight. Heifer was slaughtered at a local abattoir 14 days after ovulation and used for this study. Ovaries were collected and shipped at room temperature to the laboratory.  Approximately 3 mm3 samples of luteal tissue were placed in cryovials and frozen at -80oC until processed.
One sample was ground to a fine powder in liquid N and total RNA was extracted using the RNeasy® Mini Kit (Qiagen, Valencia, CA).  Quality and quantity of RNA was determined by gel electrophoresis and spectrophotometry.
Five µg of total RNA was converted to cDNA and then biotin-labeled cRNA by linear amplification (CodeLink® Expression Bioarrays, Amersham Biosciences Corp, Piscataway, NJ, USA). Ten micrograms of labeled cRNA were hybridized to CodeLink UniSet Rat I Bioarray (Amersham Biosciences Corp, Piscataway, NJ, USA) by the Amersham Biosciences Facility.

source_name: Ovary (luteal tissue); EID3-T00166524


title: Endophyte free, rep 3

description:
Gene expression analysis in heifer luteal tissue by interspecies microarray analysis.
Post-pubertal heifer fed diet containing endophyte free fescue seed. Heifer was slaughtered at a local abattoir 11 days after ovulation and used for this study. Ovaries were collected and shipped at room temperature to the laboratory.  Approximately 3 mm3 samples of luteal tissue were placed in cryovials and frozen at -80oC until processed.
Two samples were ground to a fine powder in liquid N and total RNA was extracted using the RNeasy® Mini Kit (Qiagen, Valencia, CA).  Quality and quantity of RNA was determined by gel electrophoresis and spectrophotometry.
Five µg of total RNA was converted to cDNA and then biotin-labeled cRNA by linear amplification (CodeLink® Expression Bioarrays, Amersham Biosciences Corp, Piscataway, NJ, USA). Ten micrograms of labeled cRNA were hybridized to CodeLink UniSet Rat I Bioarray (Amersham Biosciences Corp, Piscataway, NJ, USA) by the Amersham Biosciences Facility.

source_name: Ovary (luteal tissue); EF3-T00166521


title: Endophyte infected + domperidone, rep 2

source_name: Ovary (luteal tissue); EID2-T00166523


title: Endophyte infected, rep 1

source_name: Ovary (luteal tissue); EI1-T00168436


title: Endophyte free, rep 1

source_name: Ovary (luteal tissue); EF1-T00166509


title: CTX066 oligoarray 3

status: Public on Oct 22 2003

city: Madison

first: Jiang

last: Li

state: WI

address: 777 Highland Ave.

department: School of Pharmacy

institute: University of Wisconsin Madison

description:
Human neural stem cells (CTX066), provided by Dr. Clive Svendsen (Waisman Center, University of Wisconsin at Madison), were generated from human embryonic cortical tissue (8 weeks post conception). The neurospheres were passaged every 14 days by sectioning into 150 µM section, which then were reseeded into fresh growth medium containing 70% DMEM, 30% Hams F12, 1% penicillin/streptomycin/amphotericin B supplemented with 20 µg/ml EGF, 10 ng/ml LIF and 1% v/v N2 at a density equivalent to 200,000 cells per ml. Half the growth medium was replenished every four days. The cells were treated with EtOH (0.01%) or tBHQ (20µM) for 24 hours and harvested for microarray analysis.

Keywords = human neural stem cells
Keywords = tert-butylhydroquinone
Keywords = antioxidant responsive element

source_name: Human neural stem cells treated with Vehicle (EtOH, 0.01%)

source_name: Human neural stem cells treated with tert-butylhydroquinone (20µM)


title: Endophyte infected + domperidone, rep 1

source_name: Ovary (luteal tissue); EID1-T00166522


title: IMR-32 oligoarray 1

description:
Human neuroblastoma cells, IMR-32, were grown in Dulbecco's modified eagle's medium (DMEM) supplemented with fetal calf serum (10%), 100 I.U./ml penicillin, and 100 mg/ml streptomycin. Cultures were maintained at 37 C in a humidified 10% CO2 atmosphere.  The cells were treated with EtOH (0.01%) or tBHQ (10µM) for 24 hours and harvested for microarray analysis.

Keywords = IMR-32 cells
Keywords = tert-butylhydroquinone
Keywords = antioxidant responsive element

source_name: Human neuroblastoma cells treated with tert-butylhydroquinone (10µM)

source_name: Human neuroblastoma cells treated with Vehicle (EtOH, 0.01%)


title: IMR-32 oligoarray 2

description:
Human neuroblastoma cells, IMR-32, were grown in Dulbecco's modified eagle's medium (DMEM) supplemented with fetal calf serum (10%), 100 I.U./ml penicillin, and 100 mg/ml streptomycin. Cultures were maintained at 37 C in a humidified 10% CO2 atmosphere. The cells were treated with EtOH (0.01%) or tBHQ (10µM) for 24 hours and harvested for microarray analysis.

Keywords = IMR-32 cells
Keywords = tert-butylhydroquinone
Keywords = antioxidant responsive element


title: CTX066 oligoarray 2


title: IMR-32 oligoarray 3


title: Endophyte free, rep 2

source_name: Ovary (luteal tissue); EF2-T00166518


title: ARPE-19 cells grown on plastic.3

status: Public on Oct 15 2003

first: James

middle: T

last: Handa

address: 600 N. Wolfe St.

department: Wilmer Eye Institute

institute: Johns Hopkins Medical Institutes

description:
I.	Exp Design
1.	Type of experiment: Comparison of native versus cultured RPE cells
2.	Experimental factors: Native RPE versus ARPE-19 cells grown on different matrices
3.	How many hybridizations in exp: 21
4.	If a common reference used for all the hybs: no
5.	Quality control steps: three independent arrays for each condition
6.	Description: The expression profile of ARPE-19 cells grown on different matrices were compared to morphologically normal native macular RPE cells that were laser capture microdissected from 3 donors.

II.	Samples used, extract prep, and labeling
1.	Biosource: Human donor globes from NDRI (63, 71, 74 years old) and ARPE-19 cells.
2.	Manipulations: Human donor globes were cryopreserved, and morphologically normal RPE cells from the macula were laser capture micodissected. ARPE-19 cells were grown on different matrices (plastic, Matrigel, collagen I, collagen IV, laminin, and fibronectin).
3.	Extract preparation: Total RNA from cells were extracted with the RNeasy kit (Qiagen) using the manufacturer’s instructions.
4.	Labeling protocol: Total RNA from cells was reverse transcribed with 33P-dCTP and 33P-dATP, and second strand cDNA was labeled with 33P-dCTP and 33P-dATP.
5.	No external controls were added.

III.	Hybridization procedures and parameters
1.	Sample, array type, batch and serial # used
2.	Hybridization protocol: Hybridization was carried out using the manufacturer’s recommendations. Arrays were prehybridized with  Microhyb solution containing denatured Cot-1 DNA and poly dA at 42oC for two hours. Hybridization was carried out at 42oC overnight using a hybridization oven set at 8-10 rpm. Arrays were washed twice at 50oC for 20 minutes using 2x SSC, 1%SDS and once at room temperature for 15 minutes using 0.5x SSC, 1%SDS.
IV.	Measurement data and specifications of data processing
1,2. Arrays were exposed to a phosphorimaging screen for 3 days and scanned at 50 mm resolution with a BioRad FX Pro-Plus phosphorimager. TIFF images from the phosphorimager were exported into ResGen Pathways 3 software for analysis.
3. Data processing: A gene was expressed if its background subtracted intensity was greater than 1.4 fold background. The data were normalized using a simple global scaling procedure, and Cluster/Treeview and Statistical Analysis of Microarrays (SAM version 1.12) programs were used for analysis.

Keywords = retinal pigment epithelium
Keywords = ARPE-19
Keywords = macula
Keywords = extracellular matrix

source_name: ARPE-19 cells


title: CTX066 oligoarray 1

description:
Human neural stem cells (CTX066), provided by Dr. Clive Svendsen (Waisman Center, University of Wisconsin at Madison), were generated from human embryonic cortical tissue (8 weeks post conception). The neurospheres were passaged every 14 days by sectioning into 150 µM section, which then were reseeded into fresh growth medium containing 70% DMEM, 30% Hams F12, 1% penicillin/streptomycin/amphotericin B supplemented with 20 µg/ml EGF, 10 ng/ml LIF and 1% v/v N2 at a density equivalent to 200,000 cells per ml. Half the growth medium was replenished every four days. The cells were treated with EtOH (0.01%) or tBHQ (20µM) for 24 hours and harvested for microarray analysis.
Keywords = human neural stem cells
Keywords = tert-butylhydroquinone
Keywords = antioxidant responsive element


title: ARPE-19 cells grown on plastic.2


title: BLI_1_20447

status: Public on Oct 17 2003

description:
Ten-microgram of pooled liver total RNA obtained from nonpregnant cycling cows was reverse transcribed in the presence of cyanine-5-fluorescent dye (Cy5)-conjugated dUTP (Amersham Pharmacia Biotech) by using Superscript II reverse transcriptase (Life Technologies, Rockville, MD) to yield Cy5 fluorescence-labeled control cDNA probe. Similarly, liver total RNA obtained from a cow on day 58 of pregnancy was separately reverse transcribed in the presence of Cy3-conjugated dUTP by using Superscript II reverse transcriptase to yield Cy3 fluorescence-labeled test cDNA probe. After a 2-h reverse transcription reaction at 42oC, RNA was degraded by addition of 1.5 µl of an alkaline solution (1N NaOH / 20 mM EDTA), and neutralized by addition of 1.5 µl 1N HCl. The labeled control cDNA probe and test cDNA probe were then mixed and washed by centrifugation with TE (Tris-EDTA, pH 7.0) first and then with TE, 20 µg Salmon sperm DNA, 20 µg polyA and 20 µg yeast RNA in a Microcon YM-30 column (Millipore, Bedford, MA). The concentrated probe mix was adjusted with TE to a volume of 24.8 µl, denatured at 100oC for 2 min, spun for a few min and made to a final hybridization volume of 31 µl with 5.27 µl 20XSSC and 0.93 µl 10% SDS and kept at room temperature until used. Before hybridization, microarray glass slides were immersed in 0.2% SDS for 2 min followed by two washes each 2 min in autoclaved Milli Q water, and subjected to blocking in 180 ml of Methyl Pyrrolidinone (Aldrich) containing 2.7 g Succinic Anhydride (WAKO, Japan) and 15.3 ml 1M Boric acid (pH 8.0) solution for 20 min. Slides were then washed with autoclaved Milli Q water, and the spotted cDNA targets (microarray) were denatured in boiling water for 2 min. The slides were dehydrated in 100% ethanol for 2 min and air dried by spinning at a low speed. The mixture of labeled probes, for example, the control (Cy5) and test (Cy3), were then applied to the microarray, placed a cover slip carefully and incubated at 65oC for 14 – 16 h in a custom slide chamber with humidity maintained by a small reservoir of 3 x SSC. In addition, 3 - 6 µl of 3 x SSC was spotted at each corner of the slide, as far away from the array as possible. After incubation for 14 – 16 h at 65oC, the slides were washed in 2 x SSC / 0.03% SDS for 2 min, followed by washing in 2 x SSC, 1 x SSC and 0.2 x SSC, each for 2 min with gentle agitation, and air dried by spinning at a low speed.
Keywords = differential liver gene expression, bovine pregnancy

source_name: Adult nonpregnant liver

source_name: Adult pregnant liver (day58)


title: 12 hr A-chip 6-11-02

source_name: IB3-1 bronchial epithelial cell line


title: BLI_1_20465

description:
Ten-microgram of pooled liver total RNA obtained from nonpregnant cycling cows was reverse transcribed in the presence of cyanine-5-fluorescent dye (Cy5)-conjugated dUTP (Amersham Pharmacia Biotech) by using Superscript II reverse transcriptase (Life Technologies, Rockville, MD) to yield Cy5 fluorescence-labeled control cDNA probe. Similarly, liver total RNA obtained on day 49 of a pregnant cow was separately reverse transcribed in the presence of Cy3-conjugated dUTP by using Superscript II reverse transcriptase to yield Cy3 fluorescence-labeled test cDNA probe. After a 2-h reverse transcription reaction at 42oC, RNA was degraded by addition of 1.5 µl of an alkaline solution (1N NaOH / 20 mM EDTA), and neutralized by addition of 1.5 µl 1N HCl. A labeled control cDNA probe and a test cDNA probe were then mixed and washed by centrifugation with TE (Tris-EDTA, pH 7.0) first and then with TE, 20 µg Salmon sperm DNA, 20 µg polyA and 20 µg yeast RNA in a Microcon YM-30 column (Millipore, Bedford, MA). The concentrated probe mix was adjusted with TE to a volume of 24.8 µl, denatured at 100oC for 2 min, spun for a few min and made to a final hybridization volume of 31 µl with 5.27 µl 20XSSC and 0.93 µl 10% SDS and kept at room temperature until used. Before hybridization, microarray glass slides were immersed in 0.2% SDS for 2 min followed by two washes each 2 min in autoclaved Milli Q water, and subjected to blocking in 180 ml of Methyl Pyrrolidinone (Aldrich) containing 2.7 g Succinic Anhydride (WAKO, Japan) and 15.3 ml 1M Boric acid (pH 8.0) solution for 20 min. Slides were then washed with autoclaved Milli Q water, and the spotted cDNA targets (microarray) were denatured in boiling water for 2 min. The slides were dehydrated in 100% ethanol for 2 min and air dried by spinning at a low speed. The mixture of labeled probes, for example, the control (Cy5) and test (Cy3), were then applied to the microarray, placed a cover slip carefully and incubated at 65oC for 14 – 16 h in a custom slide chamber with humidity maintained by a small reservoir of 3 x SSC. In addition, 3 - 6 µl of 3 x SSC was spotted at each corner of the slide, as far away from the array as possible. After incubation for 14 – 16 h at 65oC, the slides were washed in 2 x SSC / 0.03% SDS for 2 min, followed by washing in 2 x SSC, 1 x SSC and 0.2 x SSC, each for 2 min with gentle agitation, and air dried by spinning at a low speed.
Keywords = differential liver gene expression, bovine pregnancy

source_name: Adult pregnant liver (day49)


title: BLI_1_20446

description:
Ten-microgram of pooled liver total RNA obtained from nonpregnant cycling cows was reverse transcribed in the presence of cyanine-5-fluorescent dye (Cy5)-conjugated dUTP (Amersham Pharmacia Biotech) by using Superscript II reverse transcriptase (Life Technologies, Rockville, MD) to yield Cy5 fluorescence-labeled control cDNA probe. Similarly, liver total RNA obtained from a cow on day 245 of pregnancy was separately reverse transcribed in the presence of Cy3-conjugated dUTP by using Superscript II reverse transcriptase to yield Cy3 fluorescence-labeled test cDNA probe. After a 2-h reverse transcription reaction at 42oC, RNA was degraded by addition of 1.5 µl of an alkaline solution (1N NaOH / 20 mM EDTA), and neutralized by addition of 1.5 µl 1N HCl. The labeled control cDNA probe and test cDNA probe were then mixed and washed by centrifugation with TE (Tris-EDTA, pH 7.0) first and then with TE, 20 µg Salmon sperm DNA, 20 µg polyA and 20 µg yeast RNA in a Microcon YM-30 column (Millipore, Bedford, MA). The concentrated probe mix was adjusted with TE to a volume of 24.8 µl, denatured at 100oC for 2 min, spun for a few min and made to a final hybridization volume of 31 µl with 5.27 µl 20XSSC and 0.93 µl 10% SDS and kept at room temperature until used. Before hybridization, microarray glass slides were immersed in 0.2% SDS for 2 min followed by two washes each 2 min in autoclaved Milli Q water, and subjected to blocking in 180 ml of Methyl Pyrrolidinone (Aldrich) containing 2.7 g Succinic Anhydride (WAKO, Japan) and 15.3 ml 1M Boric acid (pH 8.0) solution for 20 min. Slides were then washed with autoclaved Milli Q water, and the spotted cDNA targets (microarray) were denatured in boiling water for 2 min. The slides were dehydrated in 100% ethanol for 2 min and air dried by spinning at a low speed. The mixture of labeled probes, for example, the control (Cy5) and test (Cy3), were then applied to the microarray, placed a cover slip carefully and incubated at 65oC for 14 – 16 h in a custom slide chamber with humidity maintained by a small reservoir of 3 x SSC. In addition, 3 - 6 µl of 3 x SSC was spotted at each corner of the slide, as far away from the array as possible. After incubation for 14 – 16 h at 65oC, the slides were washed in 2 x SSC / 0.03% SDS for 2 min, followed by washing in 2 x SSC, 1 x SSC and 0.2 x SSC, each for 2 min with gentle agitation, and air dried by spinning at a low speed.
Keywords = differential liver gene expression, bovine pregnancy

source_name: Adult pregnant liver (day245)


title: Endophyte infected, rep 2

source_name: Ovary (luteal tissue); EI2-T00168437


title: BLI_1_20454

description:
Ten-microgram of pooled liver total RNA obtained from nonpregnant cycling cows was reverse transcribed in the presence of cyanine-5-fluorescent dye (Cy5)-conjugated dUTP (Amersham Pharmacia Biotech) by using Superscript II reverse transcriptase (Life Technologies, Rockville, MD) to yield Cy5 fluorescence-labeled control cDNA probe. Similarly, liver total RNA obtained on day 27 of a pregnant cow was separately reverse transcribed in the presence of Cy3-conjugated dUTP by using Superscript II reverse transcriptase to yield Cy3 fluorescence-labeled test cDNA probe. After a 2-h reverse transcription reaction at 42oC, RNA was degraded by addition of 1.5 µl of an alkaline solution (1N NaOH / 20 mM EDTA), and neutralized by addition of 1.5 µl 1N HCl. A labeled control cDNA probe and a test cDNA probe were then mixed and washed by centrifugation with TE (Tris-EDTA, pH 7.0) first and then with TE, 20 µg Salmon sperm DNA, 20 µg polyA and 20 µg yeast RNA in a Microcon YM-30 column (Millipore, Bedford, MA). The concentrated probe mix was adjusted with TE to a volume of 24.8 µl, denatured at 100oC for 2 min, spun for a few min and made to a final hybridization volume of 31 µl with 5.27 µl 20XSSC and 0.93 µl 10% SDS and kept at room temperature until used. Before hybridization, microarray glass slides were immersed in 0.2% SDS for 2 min followed by two washes each 2 min in autoclaved Milli Q water, and subjected to blocking in 180 ml of Methyl Pyrrolidinone (Aldrich) containing 2.7 g Succinic Anhydride (WAKO, Japan) and 15.3 ml 1M Boric acid (pH 8.0) solution for 20 min. Slides were then washed with autoclaved Milli Q water, and the spotted cDNA targets (microarray) were denatured in boiling water for 2 min. The slides were dehydrated in 100% ethanol for 2 min and air dried by spinning at a low speed. The mixture of labeled probes, for example, the control (Cy5) and test (Cy3), were then applied to the microarray, placed a cover slip carefully and incubated at 65oC for 14 – 16 h in a custom slide chamber with humidity maintained by a small reservoir of 3 x SSC. In addition, 3 - 6 µl of 3 x SSC was spotted at each corner of the slide, as far away from the array as possible. After incubation for 14 – 16 h at 65oC, the slides were washed in 2 x SSC / 0.03% SDS for 2 min, followed by washing in 2 x SSC, 1 x SSC and 0.2 x SSC, each for 2 min with gentle agitation, and air dried by spinning at a low speed.
Keywords = differential liver gene expression, bovine pregnancy

source_name: Adult pregnant liver (day27)


title: BLI_1_20456

description:
Ten-microgram of pooled liver total RNA obtained from nonpregnant cycling cows was reverse transcribed in the presence of cyanine-3-fluorescent dye (Cy3)-conjugated dUTP (Amersham Pharmacia Biotech) by using Superscript II reverse transcriptase (Life Technologies, Rockville, MD) to yield Cy3 fluorescence-labeled control cDNA probe. Similarly, liver total RNA obtained on day 49 of a pregnant cow was separately reverse transcribed in the presence of Cy5-conjugated dUTP by using Superscript II reverse transcriptase to yield Cy5 fluorescence-labeled test cDNA probe. After a 2-h reverse transcription reaction at 42oC, RNA was degraded by addition of 1.5 µl of an alkaline solution (1N NaOH / 20 mM EDTA), and neutralized by addition of 1.5 µl 1N HCl. A labeled control cDNA probe and a test cDNA probe were then mixed and washed by centrifugation with TE (Tris-EDTA, pH 7.0) first and then with TE, 20 µg Salmon sperm DNA, 20 µg polyA and 20 µg yeast RNA in a Microcon YM-30 column (Millipore, Bedford, MA). The concentrated probe mix was adjusted with TE to a volume of 24.8 µl, denatured at 100oC for 2 min, spun for a few min and made to a final hybridization volume of 31 µl with 5.27 µl 20XSSC and 0.93 µl 10% SDS and kept at room temperature until used. Before hybridization, microarray glass slides were immersed in 0.2% SDS for 2 min followed by two washes each 2 min in autoclaved Milli Q water, and subjected to blocking in 180 ml of Methyl Pyrrolidinone (Aldrich) containing 2.7 g Succinic Anhydride (WAKO, Japan) and 15.3 ml 1M Boric acid (pH 8.0) solution for 20 min. Slides were then washed with autoclaved Milli Q water, and the spotted cDNA targets (microarray) were denatured in boiling water for 2 min. The slides were dehydrated in 100% ethanol for 2 min and air dried by spinning at a low speed. The mixture of labeled probes, for example, the control (Cy3) and test (Cy5), were then applied to the microarray, placed a cover slip carefully and incubated at 65oC for 14 – 16 h in a custom slide chamber with humidity maintained by a small reservoir of 3 x SSC. In addition, 3 - 6 µl of 3 x SSC was spotted at each corner of the slide, as far away from the array as possible. After incubation for 14 – 16 h at 65oC, the slides were washed in 2 x SSC / 0.03% SDS for 2 min, followed by washing in 2 x SSC, 1 x SSC and 0.2 x SSC, each for 2 min with gentle agitation, and air dried by spinning at a low speed.
Keywords = differential liver gene expression, bovine pregnancy


title: BLI_1_20462

description:
Ten-microgram of pooled liver total RNA obtained from nonpregnant cycling cows was reverse transcribed in the presence of cyanine-5-fluorescent dye (Cy5)-conjugated dUTP (Amersham Pharmacia Biotech) by using Superscript II reverse transcriptase (Life Technologies, Rockville, MD) to yield Cy5 fluorescence-labeled control cDNA probe. Similarly, liver total RNA obtained from a cow on day 150 of pregnancy was separately reverse transcribed in the presence of Cy3-conjugated dUTP by using Superscript II reverse transcriptase to yield Cy3 fluorescence-labeled test cDNA probe. After a 2-h reverse transcription reaction at 42oC, RNA was degraded by addition of 1.5 µl of an alkaline solution (1N NaOH / 20 mM EDTA), and neutralized by addition of 1.5 µl 1N HCl. The labeled control cDNA probe and test cDNA probe were then mixed and washed by centrifugation with TE (Tris-EDTA, pH 7.0) first and then with TE, 20 µg Salmon sperm DNA, 20 µg polyA and 20 µg yeast RNA in a Microcon YM-30 column (Millipore, Bedford, MA). The concentrated probe mix was adjusted with TE to a volume of 24.8 µl, denatured at 100oC for 2 min, spun for a few min and made to a final hybridization volume of 31 µl with 5.27 µl 20XSSC and 0.93 µl 10% SDS and kept at room temperature until used. Before hybridization, microarray glass slides were immersed in 0.2% SDS for 2 min followed by two washes each 2 min in autoclaved Milli Q water, and subjected to blocking in 180 ml of Methyl Pyrrolidinone (Aldrich) containing 2.7 g Succinic Anhydride (WAKO, Japan) and 15.3 ml 1M Boric acid (pH 8.0) solution for 20 min. Slides were then washed with autoclaved Milli Q water, and the spotted cDNA targets (microarray) were denatured in boiling water for 2 min. The slides were dehydrated in 100% ethanol for 2 min and air dried by spinning at a low speed. The mixture of labeled probes, for example, the control (Cy5) and test (Cy3), were then applied to the microarray, placed a cover slip carefully and incubated at 65oC for 14 – 16 h in a custom slide chamber with humidity maintained by a small reservoir of 3 x SSC. In addition, 3 - 6 µl of 3 x SSC was spotted at each corner of the slide, as far away from the array as possible. After incubation for 14 – 16 h at 65oC, the slides were washed in 2 x SSC / 0.03% SDS for 2 min, followed by washing in 2 x SSC, 1 x SSC and 0.2 x SSC, each for 2 min with gentle agitation, and air dried by spinning at a low speed.
Keywords = differential liver gene expression, bovine pregnancy

source_name: Adult pregnant liver (day150)


title: BLI_1_20457

description:
Ten-microgram of pooled liver total RNA obtained from nonpregnant cycling cows was reverse transcribed in the presence of cyanine-3-fluorescent dye (Cy3)-conjugated dUTP (Amersham Pharmacia Biotech) by using Superscript II reverse transcriptase (Life Technologies, Rockville, MD) to yield Cy3 fluorescence-labeled control cDNA probe. Similarly, liver total RNA obtained from a cow on day 245 of pregnancy was separately reverse transcribed in the presence of Cy5-conjugated dUTP by using Superscript II reverse transcriptase to yield Cy5 fluorescence-labeled test cDNA probe. After a 2-h reverse transcription reaction at 42oC, RNA was degraded by addition of 1.5 µl of an alkaline solution (1N NaOH / 20 mM EDTA), and neutralized by addition of 1.5 µl 1N HCl. The labeled control cDNA probe and test cDNA probe were then mixed and washed by centrifugation with TE (Tris-EDTA, pH 7.0) first and then with TE, 20 µg Salmon sperm DNA, 20 µg polyA and 20 µg yeast RNA in a Microcon YM-30 column (Millipore, Bedford, MA). The concentrated probe mix was adjusted with TE to a volume of 24.8 µl, denatured at 100oC for 2 min, spun for a few min and made to a final hybridization volume of 31 µl with 5.27 µl 20XSSC and 0.93 µl 10% SDS and kept at room temperature until used. Before hybridization, microarray glass slides were immersed in 0.2% SDS for 2 min followed by two washes each 2 min in autoclaved Milli Q water, and subjected to blocking in 180 ml of Methyl Pyrrolidinone (Aldrich) containing 2.7 g Succinic Anhydride (WAKO, Japan) and 15.3 ml 1M Boric acid (pH 8.0) solution for 20 min. Slides were then washed with autoclaved Milli Q water, and the spotted cDNA targets (microarray) were denatured in boiling water for 2 min. The slides were dehydrated in 100% ethanol for 2 min and air dried by spinning at a low speed. The mixture of labeled probes, for example, the control (Cy3) and test (Cy5), were then applied to the microarray, placed a cover slip carefully and incubated at 65oC for 14 – 16 h in a custom slide chamber with humidity maintained by a small reservoir of 3 x SSC. In addition, 3 - 6 µl of 3 x SSC was spotted at each corner of the slide, as far away from the array as possible. After incubation for 14 – 16 h at 65oC, the slides were washed in 2 x SSC / 0.03% SDS for 2 min, followed by washing in 2 x SSC, 1 x SSC and 0.2 x SSC, each for 2 min with gentle agitation, and air dried by spinning at a low speed.
Keywords = differential liver gene expression, bovine pregnancy


title: BLI_1_20464

description:
Ten-microgram of pooled liver total RNA obtained from nonpregnant cycling cows was reverse transcribed in the presence of cyanine-3-fluorescent dye (Cy3)-conjugated dUTP (Amersham Pharmacia Biotech) by using Superscript II reverse transcriptase (Life Technologies, Rockville, MD) to yield Cy3 fluorescence-labeled control cDNA probe. Similarly, liver total RNA obtained on day 19 of a pregnant cow was separately reverse transcribed in the presence of Cy5-conjugated dUTP by using Superscript II reverse transcriptase to yield Cy5 fluorescence-labeled test cDNA probe. After a 2-h reverse transcription reaction at 42oC, RNA was degraded by addition of 1.5 µl of an alkaline solution (1N NaOH / 20 mM EDTA), and neutralized by addition of 1.5 µl 1N HCl. A labeled control cDNA probe and a test cDNA probe were then mixed and washed by centrifugation with TE (Tris-EDTA, pH 7.0) first and then with TE, 20 µg Salmon sperm DNA, 20 µg polyA and 20 µg yeast RNA in a Microcon YM-30 column (Millipore, Bedford, MA). The concentrated probe mix was adjusted with TE to a volume of 24.8 µl, denatured at 100oC for 2 min, spun for a few min and made to a final hybridization volume of 31 µl with 5.27 µl 20XSSC and 0.93 µl 10% SDS and kept at room temperature until used. Before hybridization, microarray glass slides were immersed in 0.2% SDS for 2 min followed by two washes each 2 min in autoclaved Milli Q water, and subjected to blocking in 180 ml of Methyl Pyrrolidinone (Aldrich) containing 2.7 g Succinic Anhydride (WAKO, Japan) and 15.3 ml 1M Boric acid (pH 8.0) solution for 20 min. Slides were then washed with autoclaved Milli Q water, and the spotted cDNA targets (microarray) were denatured in boiling water for 2 min. The slides were dehydrated in 100% ethanol for 2 min and air dried by spinning at a low speed. The mixture of labeled probes, for example, the control (Cy3) and test (Cy5), were then applied to the microarray, placed a cover slip carefully and incubated at 65oC for 14 – 16 h in a custom slide chamber with humidity maintained by a small reservoir of 3 x SSC. In addition, 3 - 6 µl of 3 x SSC was spotted at each corner of the slide, as far away from the array as possible. After incubation for 14 – 16 h at 65oC, the slides were washed in 2 x SSC / 0.03% SDS for 2 min, followed by washing in 2 x SSC, 1 x SSC and 0.2 x SSC, each for 2 min with gentle agitation, and air dried by spinning at a low speed.
Keywords = liver gene expression, bovine pregnancy

source_name: Adult pregnant liver (day19)


title: BLI_1_20466


title: BLI_1_20461

description:
Ten-microgram of pooled liver total RNA obtained from nonpregnant cycling cows was reverse transcribed in the presence of cyanine-3-fluorescent dye (Cy3)-conjugated dUTP (Amersham Pharmacia Biotech) by using Superscript II reverse transcriptase (Life Technologies, Rockville, MD) to yield Cy3 fluorescence-labeled control cDNA probe. Similarly, liver total RNA obtained from a cow on day 58 of pregnancy was separately reverse transcribed in the presence of Cy5-conjugated dUTP by using Superscript II reverse transcriptase to yield Cy5 fluorescence-labeled test cDNA probe. After a 2-h reverse transcription reaction at 42oC, RNA was degraded by addition of 1.5 µl of an alkaline solution (1N NaOH / 20 mM EDTA), and neutralized by addition of 1.5 µl 1N HCl. The labeled control cDNA probe and test cDNA probe were then mixed and washed by centrifugation with TE (Tris-EDTA, pH 7.0) first and then with TE, 20 µg Salmon sperm DNA, 20 µg polyA and 20 µg yeast RNA in a Microcon YM-30 column (Millipore, Bedford, MA). The concentrated probe mix was adjusted with TE to a volume of 24.8 µl, denatured at 100oC for 2 min, spun for a few min and made to a final hybridization volume of 31 µl with 5.27 µl 20XSSC and 0.93 µl 10% SDS and kept at room temperature until used. Before hybridization, microarray glass slides were immersed in 0.2% SDS for 2 min followed by two washes each 2 min in autoclaved Milli Q water, and subjected to blocking in 180 ml of Methyl Pyrrolidinone (Aldrich) containing 2.7 g Succinic Anhydride (WAKO, Japan) and 15.3 ml 1M Boric acid (pH 8.0) solution for 20 min. Slides were then washed with autoclaved Milli Q water, and the spotted cDNA targets (microarray) were denatured in boiling water for 2 min. The slides were dehydrated in 100% ethanol for 2 min and air dried by spinning at a low speed. The mixture of labeled probes, for example, the control (Cy3) and test (Cy5), were then applied to the microarray, placed a cover slip carefully and incubated at 65oC for 14 – 16 h in a custom slide chamber with humidity maintained by a small reservoir of 3 x SSC. In addition, 3 - 6 µl of 3 x SSC was spotted at each corner of the slide, as far away from the array as possible. After incubation for 14 – 16 h at 65oC, the slides were washed in 2 x SSC / 0.03% SDS for 2 min, followed by washing in 2 x SSC, 1 x SSC and 0.2 x SSC, each for 2 min with gentle agitation, and air dried by spinning at a low speed.
Keywords = differential liver gene expression, bovine pregnancy


title: BLI_1_20463

description:
Ten-microgram of pooled liver total RNA obtained from nonpregnant cycling cows was reverse transcribed in the presence of cyanine-3-fluorescent dye (Cy3)-conjugated dUTP (Amersham Pharmacia Biotech) by using Superscript II reverse transcriptase (Life Technologies, Rockville, MD) to yield Cy3 fluorescence-labeled control cDNA probe. Similarly, liver total RNA obtained from a cow on day 150 of pregnancy was separately reverse transcribed in the presence of Cy5-conjugated dUTP by using Superscript II reverse transcriptase to yield Cy5 fluorescence-labeled test cDNA probe. After a 2-h reverse transcription reaction at 42oC, RNA was degraded by addition of 1.5 µl of an alkaline solution (1N NaOH / 20 mM EDTA), and neutralized by addition of 1.5 µl 1N HCl. The labeled control cDNA probe and test cDNA probe were then mixed and washed by centrifugation with TE (Tris-EDTA, pH 7.0) first and then with TE, 20 µg Salmon sperm DNA, 20 µg polyA and 20 µg yeast RNA in a Microcon YM-30 column (Millipore, Bedford, MA). The concentrated probe mix was adjusted with TE to a volume of 24.8 µl, denatured at 100oC for 2 min, spun for a few min and made to a final hybridization volume of 31 µl with 5.27 µl 20XSSC and 0.93 µl 10% SDS and kept at room temperature until used. Before hybridization, microarray glass slides were immersed in 0.2% SDS for 2 min followed by two washes each 2 min in autoclaved Milli Q water, and subjected to blocking in 180 ml of Methyl Pyrrolidinone (Aldrich) containing 2.7 g Succinic Anhydride (WAKO, Japan) and 15.3 ml 1M Boric acid (pH 8.0) solution for 20 min. Slides were then washed with autoclaved Milli Q water, and the spotted cDNA targets (microarray) were denatured in boiling water for 2 min. The slides were dehydrated in 100% ethanol for 2 min and air dried by spinning at a low speed. The mixture of labeled probes, for example, the control (Cy3) and test (Cy5), were then applied to the microarray, placed a cover slip carefully and incubated at 65oC for 14 – 16 h in a custom slide chamber with humidity maintained by a small reservoir of 3 x SSC. In addition, 3 - 6 µl of 3 x SSC was spotted at each corner of the slide, as far away from the array as possible. After incubation for 14 – 16 h at 65oC, the slides were washed in 2 x SSC / 0.03% SDS for 2 min, followed by washing in 2 x SSC, 1 x SSC and 0.2 x SSC, each for 2 min with gentle agitation, and air dried by spinning at a low speed.
Keywords = differential liver gene expression, bovine pregnancy


title: ARPE-19 cells grown on Matrigel.1


title: Native macular RPE.2

source_name: Native macular RPE cells


title: Native macular RPE.3


title: ARPE-19 cells grown on Matrigel.3


title: ARPE-19 cells grown on laminin.1

description:
I.	Exp Design
1.	Type of experiment: Comparison of native versus cultured RPE cells
2.	Experimental factors: Native RPE versus ARPE-19 cells grown on different matrices
3.	How many hybridizations in exp: 21
4.	If a common reference used for all the hybs: no
5.	Quality control steps: three independent arrays for each condition
6.	Description: The expression profile of ARPE-19 cells grown on different matrices were compared to morphologically normal native macular RPE cells that were laser capture microdissected from 3 donors.

II.	Samples used, extract prep, and labeling
1.	Biosource: Human donor globes from NDRI (63, 71, 74 years old) and ARPE-19 cells.
2.	Manipulations: Human donor globes were cryopreserved, and morphologically normal RPE cells from the macula were laser capture micodissected. ARPE-19 cells were grown on different matrices (plastic, Matrigel, collagen I, collagen IV, laminin, and fibronectin).
3.	Extract preparation: Total RNA from cells were extracted with the RNeasy kit (Qiagen) using the manufacturer’s instructions.
4.	Labeling protocol: Total RNA from cells was reverse transcribed with 33P-dCTP and 33P-dATP, and second strand cDNA was labeled with 33P-dCTP and 33P-dATP.
5.	No external controls were added.

III.	Hybridization procedures and parameters
1.	Sample, array type, batch and serial # used
2.	Hybridization protocol: Hybridization was carried out using the manufacturer’s recommendations. Arrays were prehybridized with  Microhyb solution containing denatured Cot-1 DNA and poly dA at 42oC for two hours. Hybridization was carried out at 42oC overnight using a hybridization oven set at 8-10 rpm. Arrays were washed twice at 50oC for 20 minutes using 2x SSC, 1%SDS and once at room temperature for 15 minutes using 0.5x SSC, 1%SDS.
IV.	Measurement data and specifications of data processing
1,2. Arrays were exposed to a phosphorimaging screen for 3 days and scanned at 50 mm resolution with a BioRad FX Pro-Plus phosphorimager. TIFF images from the phosphorimager were exported into ResGen Pathways 3 software for analysis.
3. Data processing: A gene was expressed if its background subtracted intensity was greater than 1.4 fold background. The data were normalized using a simple global scaling procedure, and Cluster/Treeview and Statistical Analysis of Microarrays (SAM version 1.12) programs were used for analysis.

Keywords = pigment epithelium
Keywords = ARPE-19
Keywords = macula
Keywords = extracellular matrix


title: Native macular RPE.1


title: ARPE-19 cells grown on laminin.3


title: ARPE-19 cells grown on laminin.2