title: Patient D

description:
Pathohistological and clinical examination of Patient D (59 year-old male) yielded no significant pathological findings.  This patient was considered as a normal control for the purposes of the study. A complete description of the RNA extraction is available under GSE405.

Target Labeling and Hybridization procedure:

100-250 ng mRNA was isolated from total RNA for each patient using the Oligotex kit (Qiagen) according to the manufacturer’s instructions.  Prior to hybridization, 20ng of Arabidopsis thaliana DNA template was labeled with a-33P dCTP (10 microCi/microL; Amersham) during a random priming labeling reaction (pd(N)6, Amersham) (Feinberg and Vogelstein, 1983).  Reversed transcribed cDNA from patient targets were prepared in a final volume of 30microL as described in detail previously, with the exception that the labeling reaction was carried out at 42°C for 50 min (Eickhoff et al. 2000).  RNA was hydrolyzed under alkaline conditions by the addition of 3.5 microL of 3M NaOH and incubation for 20 min at 68°C. The samples were neutralized with the addition of 10 microL of 1M Tris-HCl (pH 7.4) and 10.5 microL of 1M HCl.  Unincorporated radioactive nucleotides were removed from both control and patient targets by filtration through Microspin G-50 columns according to the product manual (Amersham).

Prior to each hybridization reaction, unused filters were initially washed in 1 X SSC; 0.1% SDS at room temperature for 10 min (X 2) followed by three washes in 0.1 X SSC; 0.1% SDS for 20 min at 80°C.  These washes eliminated the potential problem for unbound or loosely bound probe cDNA from interfering with the hybridization reaction.  Each cDNA filter was pre-wetted with deionized water and placed in a hybridization bottle (3.5 X 25 cm).  The water was completely poured off and the filter was pre-hybridized for 2 hrs at 50°C in a 10 mL of hybridization solution containing 7% SDS, 50% formamide, 5 X SCC, 2% blocking reagent (Roche Diagnostics), 50mM sodium phosphate (pH 7.0) and denatured herring sperm DNA (50microg/microL).  The labeled target was added to the filters in 5 mL of the same preheated solution and hybridization of the filters was performed overnight (24 hrs) at 42°C.  Following overnight hybridization, membranes were removed from the bottles and washed in 1 X SSC/0.1% SDS buffer for 10 min at room temperature followed by two washes for 30 min in 0.2 X SSC/0.1% SDS at 65°C.  Excess moisture was removed by briefly placing the membranes on filter paper (Whatman) and the damp filters were wrapped in commercial grade plastic wrap.  Filters were exposed ~24 hrs to imaging plates (BAS-MS 2325, Fujifilm, Japan) and scanning was carried out at a resolution of 50microns on a FLA-3000G imaging system (Fujifilm, Japan). After hybridization, filters were individually stripped in a boiling solution of 0.5% SDS for 5 min, taken off the heat and agitated for an additional 3 hrs at room temperature and stored damp wrapped in plastic wrap at –20ºC for 7-8 weeks (half-life of 33P is 25.4 days). Prior to re-use, filters were re-scanned as above to ensure that residual radioactivity was negligible. For the purposes of the microarray comparison study (GSM5994-5998), all filters were chosen from the same spotting production run and all filters were used for the first time.

Spot quantitation:
An exposure time of ~24 h was sufficient to yield signals from cDNA clones, yet keeping the data overflow to a mimimum.  Images were quantified using VisualGrid® software V3.4 (http://www.gpc-biotech.com). Each grid was manually adjusted to the image such that the linear correlation coefficient between duplicate spots was greater than 0.984. The following output values from VisualGrid were used for subsequent data analysis: SIGNAL_RAW1 (the weighted mean pixel intensity within the node mask for the first duplicate), SIGNAL_RAW2 (the weighted mean pixel intensity within the node mask for the second duplicate), BKD_MEAN (the mean of the weighted means of all background spots in a 6 X 6 block).  For this dataset (GSM5994-5998), the weight of each pixel was set to one.

References:
Eickhoff, H. et al. Tissue gene expression analysis using arrayed normalized cDNA libraries. Genome Res 10, 1230-40. (2000).
Feinberg, A.P. & Vogelstein, B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132, 6-13. (1983).


title: Adult1_b

status: Public on Dec 31 2003

type: RNA

city: Heidelberg

first: Boris

last: Beckmann

address: Im Neuenheimer Feld 580

department: Funktionelle Genomanalyse

country: Germany

institute: Deutsches Krebsforschungszentrum

description:
Cy5/Cy3 labeled RNA was co-hybridized onto a QMT Amino (Quantifoil) glass slide. Embryo 0-4 h was used as common reference control.
Sample collection:
Embryo samples were collected as four-hour egg lays, which were allowed to develop for the desired interval
and then snap frozen in liquid nitrogen.
The different larval stages as well as the different pupal stages were handpicked and separately snap frozen.
Adults were collected as male and female flies and also snap frozen. Total RNA was isolated from all samples
using the Trizol reagent (Invitrogen, Karlsruhe, Germany).
Microarray hybridization:
At least three independent experiments were performed for each of the 8 different stages
(embryonic stage 4-8h, embryonic stage 8-12h, embryonic stage 12-16h, pooled larval stage, pupal stage 1, pupal stage 2,
pupal stage 3 and adult stage) in competitive hybridizations against the common control,
which was a sample made from RNA isolated from embryonic stage 0-4h. At least one dye swap was included in each of the repetitions.
We used the indirect labeling method, with 9µg random hexamer primer (Invitrogen, Karlsruhe, Germany) added to 20µg total RNA.
For hybridization, the labeled cDNAs were taken up in SlideHyb buffer #1 (Ambion, Woodward, USA), denatured at 95°C for 5min,
and applied to the array. Hybridization was performed in appropriate chambers (TeleChem) in a waterbath at 55°C for 16h.
The hybridized glass slides were scanned on a ScanArray 5000 (Perkin Elmer, Wellesley, USA) using the ScanArray software (Version 3.1).
The resulting images (TIFF format, 16-bit grayscale) for each channel - Cy5 (632nm) and Cy3 (532nm) - were analyzed
further with the GenePix software (Version 4.0; Axon Instruments, Union City, USA).
As each microarray contains two replicates for each gene, we used the standard deviation (SD)
of their dye-ratio (Cy5/Cy3) to filter for reproducible hybridizations. Only if at least 30% of all genes on the array
had a SD of log(632/532) of less than 1/3 between the replicates was the microarray included for further analysis.

molecule: total RNA

organism: Drosophila melanogaster

source_name: total RNA from young adult fly labeled with Cy3 (532 nm)

source_name: total RNA from embryo 0-4 h labeled with Cy5 (632 nm)


title: Larval_b

source_name: total RNA from larvae labeled with Cy3 (532 nm)


title: embryo_8_12_c

source_name: total RNA from embryo 8-12 h labeled with Cy5 (632 nm)

source_name: total RNA from embryo 0-4 h labeled with Cy3 (532 nm)


title: Adult1_c


title: I-7_POST

status: Public on Jan 17 2004

city: Minneapolis

first: Grindle

middle: M

last: Suzanne

state: MN

address: Dwan CVRC 289, MMC 508, 420 Delaware Street SE

department: Division of Cardiology

country: USA

institute: Lillehei Heart Institute

description:
19 paired human left ventricular apex samples were harvested at the time of implant of a left ventricular assist device (PRE) and at the time of explant (POST). The cohort included patients that were clinically classified as "ischemic" (I) showing evidence of coronary artery disease, "non-ischemic" (N) no evidence of coronary artery disease or "acute Myocardial  infarction" (IM) myocardial infarction within 10 days of the implant. Tissue was processed and hybridized to the Affymetrix HG-U133A chip.
Keywords = Heart, Cardiac, Assist Device, LVAD, GATA-4, Sprouty1, microarray, heart failure

organism: Homo sapiens

source_name: Left Cardiac Ventricle


title: Patient D (U95A)

status: Public on Dec 14 2003

city: Kiel

first: Nancy

last: Mah

institute: Christian-Albrechts-University Kiel

description:
Pathohistological and clinical examination of Patient D (59 year-old male) yielded no significant pathological findings.  This patient was considered as a normal control for the purposes of the study.
Total RNA from each sample was used to prepare biotinylated target RNA, according to the manufacturer’s protocol (http://www.affymetrix.com/support/technical/manual/expression_manual.affx). Briefly, 15 microg of total RNA was used to generate double-stranded cDNA using Superscript reagents (Life Technologies) and a T7-linked oligo(dT) primer. cRNA were synthesized using the Enzo Bioarray High Yield RNA transcript labeling kit from Affymetrix, resulting in biotinylated cRNA. Labeled cRNA were cleaned using Qiagen RNeasy kit and fragmented into 35-200 bp lengths using fragmentation buffer supplied by Affymetrix. Spike controls B2, bio-B, bio-C, bio-D and Cre-x were added to the hybridization cocktail before overnight hybridization at 45°C for 16 hrs. Arrays were stained and washed using the EukGE-WS2 protocol (a dual staining) before being scanned on an Affymetrix scanner. Absolute analysis (Affymetrix Microarray Analysis Suite 5.0) was performed in the arrays using the target intensity value of 150 (global scaling, no baseline), resulting in a 7.616 scaling factor for this sample.

source_name: sigmoid colon biopsy


title: Pupal2_c

source_name: total RNA from pupal 2 labeled with Cy5 (632 nm)


title: embryo_12_16_b

source_name: total RNA from embryo 12-16 h labeled with Cy3 (532 nm)


title: Pupal3_a

source_name: total RNA from pupal 3 labeled with Cy5 (632 nm)


title: Pupal2_b

source_name: total RNA from pupal 2 labeled with Cy3 (532 nm)


title: Pupal3_c

source_name: total RNA from pupal 3 labeled with Cy3 (532 nm)


title: embryo_12_16_a


title: embryo_8_12_b

source_name: total RNA from embryo 8-12 h labeled with Cy3 (532 nm)


title: Patient C

description:
Pathohistological and clinical examination of Patient C (62 year-old female) yielded no significant pathological findings.  This patient was considered as a normal control for the purposes of the study.
A complete description of the RNA extraction is available under GSE405.

Target Labeling and Hybridization procedure:

100-250 ng mRNA was isolated from total RNA for each patient using the Oligotex kit (Qiagen) according to the manufacturer’s instructions.  Prior to hybridization, 20ng of Arabidopsis thaliana DNA template was labeled with a-33P dCTP (10 microCi/microL; Amersham) during a random priming labeling reaction (pd(N)6, Amersham) (Feinberg and Vogelstein, 1983).  Reversed transcribed cDNA from patient targets were prepared in a final volume of 30microL as described in detail previously, with the exception that the labeling reaction was carried out at 42°C for 50 min (Eickhoff et al. 2000).  RNA was hydrolyzed under alkaline conditions by the addition of 3.5 microL of 3M NaOH and incubation for 20 min at 68°C. The samples were neutralized with the addition of 10 microL of 1M Tris-HCl (pH 7.4) and 10.5 microL of 1M HCl.  Unincorporated radioactive nucleotides were removed from both control and patient targets by filtration through Microspin G-50 columns according to the product manual (Amersham).

Prior to each hybridization reaction, unused filters were initially washed in 1 X SSC; 0.1% SDS at room temperature for 10 min (X 2) followed by three washes in 0.1 X SSC; 0.1% SDS for 20 min at 80°C.  These washes eliminated the potential problem for unbound or loosely bound probe cDNA from interfering with the hybridization reaction.  Each cDNA filter was pre-wetted with deionized water and placed in a hybridization bottle (3.5 X 25 cm).  The water was completely poured off and the filter was pre-hybridized for 2 hrs at 50°C in a 10 mL of hybridization solution containing 7% SDS, 50% formamide, 5 X SCC, 2% blocking reagent (Roche Diagnostics), 50mM sodium phosphate (pH 7.0) and denatured herring sperm DNA (50microg/microL).  The labeled target was added to the filters in 5 mL of the same preheated solution and hybridization of the filters was performed overnight (24 hrs) at 42°C.  Following overnight hybridization, membranes were removed from the bottles and washed in 1 X SSC/0.1% SDS buffer for 10 min at room temperature followed by two washes for 30 min in 0.2 X SSC/0.1% SDS at 65°C.  Excess moisture was removed by briefly placing the membranes on filter paper (Whatman) and the damp filters were wrapped in commercial grade plastic wrap.  Filters were exposed ~24 hrs to imaging plates (BAS-MS 2325, Fujifilm, Japan) and scanning was carried out at a resolution of 50microns on a FLA-3000G imaging system (Fujifilm, Japan). After hybridization, filters were individually stripped in a boiling solution of 0.5% SDS for 5 min, taken off the heat and agitated for an additional 3 hrs at room temperature and stored damp wrapped in plastic wrap at –20ºC for 7-8 weeks (half-life of 33P is 25.4 days). Prior to re-use, filters were re-scanned as above to ensure that residual radioactivity was negligible. For the purposes of the microarray comparison study (GSM5994-5998), all filters were chosen from the same spotting production run and all filters were used for the first time.

Spot quantitation:
An exposure time of ~24 h was sufficient to yield signals from cDNA clones, yet keeping the data overflow to a mimimum.  Images were quantified using VisualGrid® software V3.4 (http://www.gpc-biotech.com). Each grid was manually adjusted to the image such that the linear correlation coefficient between duplicate spots was greater than 0.984. The following output values from VisualGrid were used for subsequent data analysis: SIGNAL_RAW1 (the weighted mean pixel intensity within the node mask for the first duplicate), SIGNAL_RAW2 (the weighted mean pixel intensity within the node mask for the second duplicate), BKD_MEAN (the mean of the weighted means of all background spots in a 6 X 6 block).  For this dataset (GSM5994-5998), the weight of each pixel was set to one.

References:
Eickhoff, H. et al. Tissue gene expression analysis using arrayed normalized cDNA libraries. Genome Res 10, 1230-40. (2000).
Feinberg, A.P. & Vogelstein, B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132, 6-13. (1983).


title: Pupal1_a

source_name: total RNA from pupal 1 labeled with Cy5 (632 nm)


title: embryo_12_16_c

source_name: total RNA from embryo 12-16 h labeled with Cy5 (632 nm)


title: Pupal2_a


title: Patient B (U95A)

description:
Pathohistological and clinical examination of Patient B (55 year-old male) yielded no significant pathological findings.  This patient was considered as a normal control for the purposes of the study.

Total RNA from each sample was used to prepare biotinylated target RNA, according to the manufacturer’s protocol (http://www.affymetrix.com/support/technical/manual/expression_manual.affx). Briefly, 15 microg of total RNA was used to generate double-stranded cDNA using Superscript reagents (Life Technologies) and a T7-linked oligo(dT) primer. cRNA were synthesized using the Enzo Bioarray High Yield RNA transcript labeling kit from Affymetrix, resulting in biotinylated cRNA. Labeled cRNA were cleaned using Qiagen RNeasy kit and fragmented into 35-200 bp lengths using fragmentation buffer supplied by Affymetrix. Spike controls B2, bio-B, bio-C, bio-D and Cre-x were added to the hybridization cocktail before overnight hybridization at 45°C for 16 hrs. Arrays were stained and washed using the EukGE-WS2 protocol (a dual staining) before being scanned on an Affymetrix scanner. Absolute analysis (Affymetrix Microarray Analysis Suite 5.0) was performed in the arrays using the target intensity value of 150 (global scaling, no baseline), resulting in a 5.38 scaling factor for this sample.


title: embryo_4_8_a

source_name: total RNA from embryo 4-8 h labeled with Cy5 (632 nm)


title: Patient A (U95A)

description:
Pathohistological and clinical examination of Patient A (70 year-old male) yielded no significant pathological findings.  This patient was considered as a normal control for the purposes of the study.

Total RNA from each sample was used to prepare biotinylated target RNA, according to the manufacturer’s protocol (http://www.affymetrix.com/support/technical/manual/expression_manual.affx). Briefly, 15 microg of total RNA was used to generate double-stranded cDNA using Superscript reagents (Life Technologies) and a T7-linked oligo(dT) primer. cRNA were synthesized using the Enzo Bioarray High Yield RNA transcript labeling kit from Affymetrix, resulting in biotinylated cRNA. Labeled cRNA were cleaned using Qiagen RNeasy kit and fragmented into 35-200 bp lengths using fragmentation buffer supplied by Affymetrix. Spike controls B2, bio-B, bio-C, bio-D and Cre-x were added to the hybridization cocktail before overnight hybridization at 45°C for 16 hrs. Arrays were stained and washed using the EukGE-WS2 protocol (a dual staining) before being scanned on an Affymetrix scanner. Absolute analysis (Affymetrix Microarray Analysis Suite 5.0) was performed in the arrays using the target intensity value of 150 (global scaling, no baseline), resulting in a 8.186 scaling factor for this sample.


title: embryo_4_8_b


title: Pupal1_b


title: Larval_a

source_name: total RNA from larvae labeled with Cy5 (632 nm)


title: Pupal1_c

source_name: total RNA from pupal 1 labeled with Cy3 (532 nm)


title: Larval_c

source_name: total RNA from embryo 0-4 h labeled with Cy5 (632 nm)


title: ANIT_24hr_3

status: Public on Dec 13 2003

city: Chiba

first: Kensuke

last: Kojima

state: Chiba

department: Graduate School of Pharmaceutical Sciences

country: Japan

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 intraperitoneally injected with 75 mg/kg body weight of ANIT (a-naphtylisothiocyanate) dissolved in olive oil (7.5 mg/mL). Control animals received an injection of the same volume of olive oil.
Groups of three rats were sacrificed under ether anesthesia 24 hr after ANIT or olive oil administration. 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 cyanine-3- and cyanine-5-dUTP (Perkin-Elmer Inc., Wellesley, MA). Poly(A) RNAs derived from rats that has administrated ANIT and from those that has administrated olive oil were labeled with cyanine-5 and cyanine-3, 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-5 and cyanine-3 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 ANIT-treatment rats and these in the samples derived from olive oil-treatment rats were calculated for each transcript and log2-transformed.

organism: Rattus norvegicus

source_name: Olive oil_Cy3_24hr


title: ANIT_24hr_4

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 intraperitoneally injected with 75 mg/kg body weight of ANIT (a-naphtylisothiocyanate) dissolved in olive oil (7.5 mg/mL). Control animals received an injection of the same volume of olive oil.
Groups of three rats were sacrificed under ether anesthesia 24 hr after ANIT or olive oil administration. 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 cyanine-3- and cyanine-5-dUTP (Perkin-Elmer Inc., Wellesley, MA). Poly(A) RNAs derived from rats that has administrated ANIT and from those that has administrated olive oil 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 ANIT-treatment rats and these in the samples derived from olive oil-treatment rats were calculated for each transcript and log2-transformed.

source_name: Olive oil_Cy5_24hr


title: Patient A

description:
Pathohistological and clinical examination of Patient A (70 year-old male) yielded no significant pathological findings.  This patient was considered as a normal control for the purposes of the study.
A complete description of the RNA extraction is available under GSE405.

Target Labeling and Hybridization procedure:

100-250 ng mRNA was isolated from total RNA for each patient using the Oligotex kit (Qiagen) according to the manufacturer’s instructions.  Prior to hybridization, 20ng of Arabidopsis thaliana DNA template was labeled with a-33P dCTP (10 microCi/microL; Amersham) during a random priming labeling reaction (pd(N)6, Amersham) (Feinberg and Vogelstein, 1983).  Reversed transcribed cDNA from patient targets were prepared in a final volume of 30microL as described in detail previously, with the exception that the labeling reaction was carried out at 42°C for 50 min (Eickhoff et al. 2000).  RNA was hydrolyzed under alkaline conditions by the addition of 3.5 microL of 3M NaOH and incubation for 20 min at 68°C. The samples were neutralized with the addition of 10 microL of 1M Tris-HCl (pH 7.4) and 10.5 microL of 1M HCl.  Unincorporated radioactive nucleotides were removed from both control and patient targets by filtration through Microspin G-50 columns according to the product manual (Amersham).

Prior to each hybridization reaction, unused filters were initially washed in 1 X SSC; 0.1% SDS at room temperature for 10 min (X 2) followed by three washes in 0.1 X SSC; 0.1% SDS for 20 min at 80°C.  These washes eliminated the potential problem for unbound or loosely bound probe cDNA from interfering with the hybridization reaction.  Each cDNA filter was pre-wetted with deionized water and placed in a hybridization bottle (3.5 X 25 cm).  The water was completely poured off and the filter was pre-hybridized for 2 hrs at 50°C in a 10 mL of hybridization solution containing 7% SDS, 50% formamide, 5 X SCC, 2% blocking reagent (Roche Diagnostics), 50mM sodium phosphate (pH 7.0) and denatured herring sperm DNA (50microg/microL).  The labeled target was added to the filters in 5 mL of the same preheated solution and hybridization of the filters was performed overnight (24 hrs) at 42°C.  Following overnight hybridization, membranes were removed from the bottles and washed in 1 X SSC/0.1% SDS buffer for 10 min at room temperature followed by two washes for 30 min in 0.2 X SSC/0.1% SDS at 65°C.  Excess moisture was removed by briefly placing the membranes on filter paper (Whatman) and the damp filters were wrapped in commercial grade plastic wrap.  Filters were exposed ~24 hrs to imaging plates (BAS-MS 2325, Fujifilm, Japan) and scanning was carried out at a resolution of 50microns on a FLA-3000G imaging system (Fujifilm, Japan). After hybridization, filters were individually stripped in a boiling solution of 0.5% SDS for 5 min, taken off the heat and agitated for an additional 3 hrs at room temperature and stored damp wrapped in plastic wrap at –20ºC for 7-8 weeks (half-life of 33P is 25.4 days). Prior to re-use, filters were re-scanned as above to ensure that residual radioactivity was negligible. For the purposes of the microarray comparison study (GSM5994-5998), all filters were chosen from the same spotting production run and all filters were used for the first time.

Spot quantitation:
An exposure time of ~24 h was sufficient to yield signals from cDNA clones, yet keeping the data overflow to a mimimum.  Images were quantified using VisualGrid® software V3.4 (http://www.gpc-biotech.com). Each grid was manually adjusted to the image such that the linear correlation coefficient between duplicate spots was greater than 0.984. The following output values from VisualGrid were used for subsequent data analysis: SIGNAL_RAW1 (the weighted mean pixel intensity within the node mask for the first duplicate), SIGNAL_RAW2 (the weighted mean pixel intensity within the node mask for the second duplicate), BKD_MEAN (the mean of the weighted means of all background spots in a 6 X 6 block).  For this dataset (GSM5994-5998), the weight of each pixel was set to one.

References:
Eickhoff, H. et al. Tissue gene expression analysis using arrayed normalized cDNA libraries. Genome Res 10, 1230-40. (2000).
Feinberg, A.P. & Vogelstein, B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132, 6-13. (1983).


title: ANIT_18hr_4

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 intraperitoneally injected with 75 mg/kg body weight of ANIT (a-naphtylisothiocyanate) dissolved in olive oil (7.5 mg/mL). Control animals received an injection of the same volume of olive oil.
Groups of three rats were sacrificed under ether anesthesia 18 hr after ANIT or olive oil administration. 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 cyanine-3- and cyanine-5-dUTP (Perkin-Elmer Inc., Wellesley, MA). Poly(A) RNAs derived from rats that has administrated ANIT and from those that has administrated olive oil 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 ANIT-treatment rats and these in the samples derived from olive oil-treatment rats were calculated for each transcript and log2-transformed.

source_name: Olive oil_Cy5_18hr


title: ANIT_18hr_1

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 intraperitoneally injected with 75 mg/kg body weight of ANIT (a-naphtylisothiocyanate) dissolved in olive oil (7.5 mg/mL). Control animals received an injection of the same volume of olive oil.
Groups of three rats were sacrificed under ether anesthesia 18 hr after ANIT or olive oil administration. 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 cyanine-3- and cyanine-5-dUTP (Perkin-Elmer Inc., Wellesley, MA). Poly(A) RNAs derived from rats that has administrated ANIT and from those that has administrated olive oil were labeled with cyanine-5 and cyanine-3, 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-5 and cyanine-3 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 ANIT-treatment rats and these in the samples derived from olive oil-treatment rats were calculated for each transcript and log2-transformed.

source_name: Olive oil_Cy3_18hr


title: ANIT_24hr_2


title: Pupal3_b

source_name: total RNA from embryo 0-4 h labeled with Cy3 (532 nm)


title: Patient B

description:
Pathohistological and clinical examination of Patient B (55 year-old male) yielded no significant pathological findings.  This patient was considered as a normal control for the purposes of the study.
A complete description of the RNA extraction is available under GSE405.

Target Labeling and Hybridization procedure:

100-250 ng mRNA was isolated from total RNA for each patient using the Oligotex kit (Qiagen) according to the manufacturer’s instructions.  Prior to hybridization, 20ng of Arabidopsis thaliana DNA template was labeled with a-33P dCTP (10 microCi/microL; Amersham) during a random priming labeling reaction (pd(N)6, Amersham) (Feinberg and Vogelstein, 1983).  Reversed transcribed cDNA from patient targets were prepared in a final volume of 30microL as described in detail previously, with the exception that the labeling reaction was carried out at 42°C for 50 min (Eickhoff et al. 2000).  RNA was hydrolyzed under alkaline conditions by the addition of 3.5 microL of 3M NaOH and incubation for 20 min at 68°C. The samples were neutralized with the addition of 10 microL of 1M Tris-HCl (pH 7.4) and 10.5 microL of 1M HCl.  Unincorporated radioactive nucleotides were removed from both control and patient targets by filtration through Microspin G-50 columns according to the product manual (Amersham).

Prior to each hybridization reaction, unused filters were initially washed in 1 X SSC; 0.1% SDS at room temperature for 10 min (X 2) followed by three washes in 0.1 X SSC; 0.1% SDS for 20 min at 80°C.  These washes eliminated the potential problem for unbound or loosely bound probe cDNA from interfering with the hybridization reaction.  Each cDNA filter was pre-wetted with deionized water and placed in a hybridization bottle (3.5 X 25 cm).  The water was completely poured off and the filter was pre-hybridized for 2 hrs at 50°C in a 10 mL of hybridization solution containing 7% SDS, 50% formamide, 5 X SCC, 2% blocking reagent (Roche Diagnostics), 50mM sodium phosphate (pH 7.0) and denatured herring sperm DNA (50microg/microL).  The labeled target was added to the filters in 5 mL of the same preheated solution and hybridization of the filters was performed overnight (24 hrs) at 42°C.  Following overnight hybridization, membranes were removed from the bottles and washed in 1 X SSC/0.1% SDS buffer for 10 min at room temperature followed by two washes for 30 min in 0.2 X SSC/0.1% SDS at 65°C.  Excess moisture was removed by briefly placing the membranes on filter paper (Whatman) and the damp filters were wrapped in commercial grade plastic wrap.  Filters were exposed ~24 hrs to imaging plates (BAS-MS 2325, Fujifilm, Japan) and scanning was carried out at a resolution of 50microns on a FLA-3000G imaging system (Fujifilm, Japan). After hybridization, filters were individually stripped in a boiling solution of 0.5% SDS for 5 min, taken off the heat and agitated for an additional 3 hrs at room temperature and stored damp wrapped in plastic wrap at –20ºC for 7-8 weeks (half-life of 33P is 25.4 days). Prior to re-use, filters were re-scanned as above to ensure that residual radioactivity was negligible. For the purposes of the microarray comparison study (GSM5994-5998), all filters were chosen from the same spotting production run and all filters were used for the first time.

Spot quantitation:
An exposure time of ~24 h was sufficient to yield signals from cDNA clones, yet keeping the data overflow to a mimimum.  Images were quantified using VisualGrid® software V3.4 (http://www.gpc-biotech.com). Each grid was manually adjusted to the image such that the linear correlation coefficient between duplicate spots was greater than 0.984. The following output values from VisualGrid were used for subsequent data analysis: SIGNAL_RAW1 (the weighted mean pixel intensity within the node mask for the first duplicate), SIGNAL_RAW2 (the weighted mean pixel intensity within the node mask for the second duplicate), BKD_MEAN (the mean of the weighted means of all background spots in a 6 X 6 block).  For this dataset (GSM5994-5998), the weight of each pixel was set to one.

References:
Eickhoff, H. et al. Tissue gene expression analysis using arrayed normalized cDNA libraries. Genome Res 10, 1230-40. (2000).
Feinberg, A.P. & Vogelstein, B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132, 6-13. (1983).

source_name: sigmoid colon biopsy


title: ANIT_24hr_1


title: Adult1_a

source_name: total RNA from young adult fly labeled with Cy5 (632 nm)


title: ANIT_18hr_2


title: ANIT_12hr_3

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 intraperitoneally injected with 75 mg/kg body weight of ANIT (a-naphtylisothiocyanate) dissolved in olive oil (7.5 mg/mL). Control animals received an injection of the same volume of olive oil.
Groups of three rats were sacrificed under ether anesthesia 12 hr after ANIT or olive oil administration. 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 cyanine-3- and cyanine-5-dUTP (Perkin-Elmer Inc., Wellesley, MA). Poly(A) RNAs derived from rats that has administrated ANIT and from those that has administrated olive oil were labeled with cyanine-5 and cyanine-3, 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-5 and cyanine-3 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 ANIT-treatment rats and these in the samples derived from olive oil-treatment rats were calculated for each transcript and log2-transformed.

source_name: Olive oil_Cy3_12hr


title: ANIT_12hr_2

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 intraperitoneally injected with 75 mg/kg body weight of ANIT (a-naphtylisothiocyanate) dissolved in olive oil (7.5 mg/mL). Control animals received an injection of the same volume of olive oil.
Groups of three rats were sacrificed under ether anesthesia 12 hr after ANIT or olive oil administration. 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 cyanine-3- and cyanine-5-dUTP (Perkin-Elmer Inc., Wellesley, MA). Poly(A) RNAs derived from rats that has administrated ANIT and from those that has administrated olive oil 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 ANIT-treatment rats and these in the samples derived from olive oil-treatment rats were calculated for each transcript and log2-transformed.

source_name: Olive oil_Cy5_12hr


title: ANIT_12hr_4


title: embryo_4_8_c

source_name: total RNA from embryo 4-8 h labeled with Cy3 (532 nm)


title: BDL_12hr_4

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 12 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.

source_name: Sham operation_Cy5_12hr

source_name: Common bile duct ligation_Cy3_12hr


title: embryo_8_12_a

source_name: total RNA from embryo 8-12 h labeled with Cy5 (632 nm)


title: BDL_12hr_2


title: BDL_12hr_1

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 12 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-5 and cyanine-3, 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-5 and cyanine-3 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.

source_name: Sham operation_Cy3_12hr

source_name: Common bile duct ligation_Cy5_12hr


title: ANIT_18hr_3


title: BDL_3hr_3

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-5 and cyanine-3, 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-5 and cyanine-3 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.

source_name: Sham operation_Cy3_3hr

source_name: Common bile duct ligation_Cy5_3hr


title: BDL_3hr_4

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.

source_name: Sham operation_Cy5_3hr

source_name: Common bile duct ligation_Cy3_3hr


title: BDL_1hr_4

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 1 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.

source_name: Sham operation_Cy5_1hr

source_name: Common bile duct ligation_Cy3_1hr


title: BDL_3hr_1


title: BDL_1hr_2


title: Patient C (U95A)

description:
Pathohistological and clinical examination of Patient C (62 year-old female) yielded no significant pathological findings.  This patient was considered as a normal control for the purposes of the study.
Total RNA from each sample was used to prepare biotinylated target RNA, according to the manufacturer’s protocol (http://www.affymetrix.com/support/technical/manual/expression_manual.affx). Briefly, 15 microg of total RNA was used to generate double-stranded cDNA using Superscript reagents (Life Technologies) and a T7-linked oligo(dT) primer. cRNA were synthesized using the Enzo Bioarray High Yield RNA transcript labeling kit from Affymetrix, resulting in biotinylated cRNA. Labeled cRNA were cleaned using Qiagen RNeasy kit and fragmented into 35-200 bp lengths using fragmentation buffer supplied by Affymetrix. Spike controls B2, bio-B, bio-C, bio-D and Cre-x were added to the hybridization cocktail before overnight hybridization at 45°C for 16 hrs. Arrays were stained and washed using the EukGE-WS2 protocol (a dual staining) before being scanned on an Affymetrix scanner. Absolute analysis (Affymetrix Microarray Analysis Suite 5.0) was performed in the arrays using the target intensity value of 150 (global scaling, no baseline), resulting in a 27.8 scaling factor for this sample.


title: BDL_1hr_1

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 1 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-5 and cyanine-3, 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-5 and cyanine-3 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.

source_name: Sham operation_Cy3 _1hr

source_name: Common bile duct ligation_Cy5 _1hr


title: BDL_1hr_3

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 1 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-5 and cyanine-3, 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-5 and cyanine-3 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.

source_name: Sham operation_Cy3_1hr

source_name: Common bile duct ligation_Cy5_1hr


title: Stationary Phase cells, time 180 seconds

status: Public on Feb 19 2005

city: Albuquerque

first: Sushmita

last: Roy

state: NM

institute: Biology, University of New Mexico

description:
Yeast cells cultured for 7 days in YPD+A subsequently exposed to 50 uM menadione
Cells were collected every 10 seconds for 3 minutes with a final time point a 5 minutes. All RNA isolation was accomplished with a modified Gentra protocol followed by Qiagen RNeasy Mini Kit reagents. As a control, exponential and stationary phase RNA were mixied 1:1. RNA (20 µg) was annealed with 2 µg oligo dT, and reverse-transcribed into cDNA with Superscript II reverse transcriptase for 2h at 42°C in the presence of 0.5 mM dGTP, 0.25 mM dCTP, 0.5 mM dATP, 0.5 mM dTTP. RNA was degraded using RNase H and RNase A.
The Cy5 and Cy3 labelled cDNAs were combined and purified with a QIAquick PCR purification kit and suspended in 30 µl of the hybridization solution.
Fluorescent array images were collected for both Cy3 and Cy5 with a GenePix 4000B fluorescent scanner and image intensity data were extracted and analyzed with GenePix Pro 3.0 analysis software. After background correction and removal of flagged values, log base 2 expression ratios were normalized to exogonus controls.
Keywords = Stationary phase yeast

organism: Saccharomyces cerevisiae

source_name: Stationary Phase and Exponental mix 1:1


title: ANIT_12hr_1

source_name: Olive oil_Cy3_12hr


title: BDL_12hr_3


title: mouse_testis_tubular_somatic_cells_GPL81

status: Public on Nov 07 2003

city: New York

first: Nikolaus

last: Schultz

state: NY

address: 1275 York Ave, Box 460

department: Computational Biology Center

institute: Memorial Sloan-Kettering Cancer Center

description:
RNA preparation was performed using RNA Stat-60 (Teltest, Friendswood, TX), according to the manufacturer’s protocol. 50 ng total RNA were amplified using two rounds of amplification. Detection calls were calculated using a Tau value of 0.015. Signal intensities were scaled to an average of 250 (scale factor 1.389).
Keywords = Mus musculus, testis spermatogenesis

organism: Mus musculus

source_name: 19 day old testis, tubular somatic cells


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

status: Public on Oct 29 2003

city: Baltimore

first: Jerry

middle: M.

last: Wright

state: MD

address: 725 N. Wolfe St

department: Physiology

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.

source_name: IB3-1 bronchial epithelial cell line


title: mouse_testis_tubular_somatic_cells_GPL82

description:
RNA preparation was performed using RNA Stat-60 (Teltest, Friendswood, TX), according to the manufacturer’s protocol. 50 ng total RNA were amplified using two rounds of amplification. Detection calls were calculated using a Tau value of 0.015. Signal intensities were scaled to an average of 250 (scale factor 4.011).
Keywords = Mus musculus, testis spermatogenesis


title: mouse_testis_tubular_somatic_cells_GPL83

description:
RNA preparation was performed using RNA Stat-60 (Teltest, Friendswood, TX), according to the manufacturer’s protocol. 50 ng total RNA were amplified using two rounds of amplification. Detection calls were calculated using a Tau value of 0.015. Signal intensities were scaled to an average of 250 (scale factor 7.036).
Keywords = Mus musculus, testis spermatogenesis


title: 3000002691

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 3-Oct-2001

source_name: C57Bl6 Thyroid Wild Type (Cy5)

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


title: 3000054129

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 24-Apr-2002


title: 3000002785

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:
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(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