title: Control 03, NC03

description:
Pathohistological and clinical examination of Patient NC03 (27 year-old male) yielded no significant pathological findings.  This patient was considered as a normal control for the purposes of the study.
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
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. ID (Location) 95A01 - 96P24 contained spotting buffer only and were flaged as empty / set to zero.
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: Control 06, NC06

status: Public on Aug 11 2005

type: RNA

city: Kiel

first: Robert

last: Haesler

address: Schittenhelmstr. 12

department: IKMB / 1st Dept. of Medicine

country: Germany

institute: University Clinic of Schleswig Holstein

description:
Pathohistological and clinical examination of Patient NC06 (57 year-old female) yielded no significant pathological findings.  This patient was considered as a normal control for the purposes of the study.
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
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. ID (Location) 95A01 - 96P24 contained spotting buffer only and were flaged as empty / set to zero.
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).

molecule: total RNA

organism: Homo sapiens

source_name: sigmoid colon biopsy


title: Crohn 06, CD06

description:
Pathohistological and clinical examination of Patient CD06 (66 year-old male) yielded to diagnosis of Crohn's disease.
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.
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. ID (Location) 95A01 - 96P24 contained spotting buffer only and were flaged as empty / set to zero.
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: IGR sir2-myc binding map(#15)

type: genomic

city: Stockholm

first: Karl

last: Ekwall

address: Alfred Nobels Alle 7

country: Sweden

institute: Karolinska Inst

description: For HDAC binding (Clr3-myc and Sir2- myc), ChIP was performed according to Kurdistani et al (2002), also with the modification to the protocol of leaving out the cold PBS wash. We used 1.5–3.0 mg 9E10 monoclonal antibody against Myc(Sigma) for each IP.

molecule: genomic DNA

organism: Schizosaccharomyces pombe

source_name: CHIP IP sir2-myc

tag: sir2-myc sample

source_name: CHIP Input

tag: CHIP input sir2-myc


title: Control 08, NC08

description:
Pathohistological and clinical examination of Patient NC08 (27 year-old female) yielded no significant pathological findings.  This patient was considered as a normal control for the purposes of the study.
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
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. ID (Location) 95A01 - 96P24 contained spotting buffer only and were flaged as empty / set to zero.
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: Crohn 07, CD07

description:
Pathohistological and clinical examination of Patient CD07 (25 year-old male) yielded to diagnosis of Crohn's disease.
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.
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. ID (Location) 95A01 - 96P24 contained spotting buffer only and were flaged as empty / set to zero.
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: Control 07, NC07

description:
Pathohistological and clinical examination of Patient NC07 (84 year-old female) yielded no significant pathological findings.  This patient was considered as a normal control for the purposes of the study.
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
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. ID (Location) 95A01 - 96P24 contained spotting buffer only and were flaged as empty / set to zero.
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: Control 10, NC10

description:
Pathohistological and clinical examination of Patient NC10 (72 year-old male) yielded no significant pathological findings.  This patient was considered as a normal control for the purposes of the study.
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
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. ID (Location) 95A01 - 96P24 contained spotting buffer only and were flaged as empty / set to zero.
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: IGR sir2-myc binding map(B160E #4)


title: Control 04, NC04

description:
Pathohistological and clinical examination of Patient NC04 (55 year-old male) yielded no significant pathological findings.  This patient was considered as a normal control for the purposes of the study.
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
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. ID (Location) 95A01 - 96P24 contained spotting buffer only and were flaged as empty / set to zero.
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: Control 05, NC05

description:
Pathohistological and clinical examination of Patient NC05 (46 year-old female) yielded no significant pathological findings.  This patient was considered as a normal control for the purposes of the study.
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
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. ID (Location) 95A01 - 96P24 contained spotting buffer only and were flaged as empty / set to zero.
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: Control 02, NC02

description:
Pathohistological and clinical examination of Patient NC02 (47 year-old female) yielded no significant pathological findings.  This patient was considered as a normal control for the purposes of the study.
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
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. ID (Location) 95A01 - 96P24 contained spotting buffer only and were flaged as empty / set to zero.
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: Crohn 08, CD08

description:
Pathohistological and clinical examination of Patient CD08 (44 year-old male) yielded to diagnosis of Crohn's disease.
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
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. ID (Location) 95A01 - 96P24 contained spotting buffer only and were flaged as empty / set to zero.
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: Crohn 05, CD05

description:
Pathohistological and clinical examination of Patient CD05 (24 year-old female) yielded to diagnosis of Crohn's disease.
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.
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. ID (Location) 95A01 - 96P24 contained spotting buffer only and were flaged as empty / set to zero.
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: hMSCsVshMSC48h3

status: Public on Aug 10 2005

city: Genoa

first: Paolo

last: Malatesta

address: Largo Rosanna Benzi 10

country: Italy

institute: IRCCS Ospedale Policlinico San Martino

description: TIFF images were analysed with TIGR Spotfinder v2.2.4. (from TIGR TM4 suite; Saeed et al., Biotechniques 34(2): 374-8. 2003) using Otsu thresholding with min SpotSize=10; maxSpotSize=55

hyb_protocol: The arrays were pre-blocked in hybridization buffer (5X SSC; 0.1%SDS; 0.2 mg/ml Torula Yeast RNA; 5X Denhardt's solution; 25% formamide) for 2 hours. 120 pmol of each dye-labeled aRNA sample, diluted in 100 µl of hybridization buffer were pre-denatured for 5 minutes at 65°C and then applied on the glass slide. A glass coverslip was used to uniformly spread the sample solution. Incubation was carried on for 24 hours at 48°C in a humid chamber and was followed by 6 washing steps with lowering concentration of SSC and SDS

scan_protocol: Array scanning was performed by using a ScanArray Lite (PerkinElmer Life Sciences) Laser Power= 75; PMT(Cy3)=85; PMT(Cy5)=80, resolution 5 µm/pixel.

data_processing: Non linear LOWESS based lockfit alogorithm employing MIDAS (from TIGR TM4 suite; Saeed et al., 2003)

source_name: Untreated Mesenchymal Stem Cells

label_protocol: Representational amplification of mRNA and fluorescent labelling was carried out using Ambion’s Amino Allyl MessageAmp aRNA kit and NHS ester Cy3 and Cy5 conjugated fluorescent dyes (Amersham Bioscience) following the protocol suggested by the supplier

extract_protocol: RNeasy Mini kit (Qiagen)

tag: Tissue

value: isolated hMSC

tag: Gender

value: male

tag: Age

value: comprised between 35 and 50

tag: Donor #3

source_name: Mesenchymal Stem Cells 48 hrs after DMSO/BHA Induction

treatment_protocol: 48 hours of exposition to DMSO/BHA induction medium as described in (Woodbury et al., J Neurosci Res 61, pp 364-370, 2000)


title: 10PGA2B

status: Public on Aug 13 2005

city: Wageningen

first: Jaap

last: Keijer

address: Bornsesteeg 45

country: Netherlands

institute: RIKILT Institute of Food Safety

description: The 3DNA Array 350 HS Expression array detection kit for microarrays (Cy3/Cy5 Kit, Genisphere, Hatfield, USA) was used for both cDNA synthesis and microarray hybridization. Briefly, 5 µg of total RNA from each sample (4 x 3) was reverse transcribed in duplo using 5 pmol capture sequence primer. The Cy5 specific primer was used for the individual samples and the Cy3 specific primer was used for the reference samples, consisting of a pool of RNA extracted from all samples. In total, 4 x 3 x 2 = 24 reactions were performed for both Cy5 and Cy3 samples. For all individual reactions, total RNA (5 µg in 1-10 µl) and RT primer (1 µl) were brought to a final volume of 11 µl with nuclease free water and incubated at 80 degrees C. Afterwards, 1 µl of Superase-In was added and a reaction mix consisting of 4 µl 5x SuperScript II first strand buffer, 1 µl dNTP mix (10 mM each for dATP, dCTP, dGTp, dTTP), 2 µl 0.1 M DTT and 1 µl Superscript II enzyme (200 units) (Invitrogen). The reaction was incubated at 42 degrees C for 2 hours. The reaction was terminated by adding 3.5 µl 0.5 M NaOH/50 mM EDTA and incubation at 65 degrees C for 10 minutes. The reaction was neutralized with 5 µl 1 M Tris-HCl, pH 7.5. The Cy5 and Cy3 reactions were then combined per sample, purified with the Qiaquick PCR purification kit (Westburg) and precipitated overnight at -20 degrees C using 1/10 volume of 3 M sodium-acetate (pH 5.2) and 2.5 volumes of 96% ethanol.

data_processing: ArrayVision for visualisation of the array, GeneMathsXT for Rikilt normalisation and further data-analysis

source_name: human H29 cell line reference

tag: total mRNA of all the experiments

source_name: human H29 cell line experiment

tag: experiment treated with 10ng/ml PGA (synthetic folic acid), second cell culture, second of the duplo


title: hMSCsVsBrain1

tag: Donor #1

source_name: Foetal brain

value: Brain

tag: Pool of 3 different samples

value: Foetal. 6 weeks


title: hMSCsVsLiver3

source_name: Adult liver

tag: Human Liver

tag: Chemicon cat. nr 562


title: hMSCsVsLiver2

tag: Donor #2

tag: Human liver

tag: Chemicon cat.Nr 562


title: hMSCsVshMSC6h3

source_name: Mesenchymal Stem Cells 6 hrs after DMSO/BHA Induction

treatment_protocol: 6 hours of exposition to DMSO/BHA induction medium as described in (Woodbury et al., J Neurosci Res 61, pp 364-370, 2000)


title: hMSCsVshMSC6h1


title: E3_T7_R3, 360 min. post rehydration

status: Public on Aug 01 2005

city: Blacksburg

first: Richard

middle: F.

last: Helm

state: VA

address: West Campus Drive

department: Biochemistry, Fralin Biotechnology Center

country: USA

institute: Virginia Polytechnic Institute and State University

description:
Stock and seed cultures of strain BY4743 were grown in yeast extract-peptone-dextrose (YPD) medium (10 mg/ml yeast extract, 20 mg/ml bactopeptone, 20 mg/ml glucose, pH 6.5 ± 0.2), at 30°C until the A600 reached 0.8.  Cells were centrifuged and the cell pellet was resuspended and dispersed in 1:20 diluted YPD medium at between 22 to 25 °C.  An aliquot, which represent the control sample (T1), was removed for RNA extraction, and the remaining suspension was aliquoted into 9-cm diameter glass Petri dishes (15 ml/dish) for desiccation.
Desiccation was carried out in a custom-fabricated controlled atmosphere desiccation system, at 22 to 25 °C.  For more information on the unit and software, contact the corresponding author.  A series of aliquots were withdrawn from the cell suspensions 18h (T2) after the start of the drying process.
Samples were collected on a volume basis and were in the same ratio as collected at T1.  Once dry (42 h), the water potential was adjusted to 20% relative humidity for a period of 72 hr. A sample was collected at this timepoint, representing the dry sample (Tdry).  Dry cells were hydrated (22 to 25 °C ) using 1:20 diluted YPD medium, 15 ml per plate. Resuspended cells from the four plates were pooled in a sterile 250 ml flask.  Samples were collected for various analyses at time points of 15 min (T4), 45 min (T5), 90 min (T6) and 360 min (T7) after the start of rehydration.
Total RNA was isolated essentially as described (Schmitt, M. E., Brown, T. A., and Trumpower, B. L. (1990) Nucl. Acids Res. 18, 3091-3092).  RNA was quantified using a spectrophotometer, and its quality was assessed using the A260/A280 ratio.
Target cRNA was hybridized to Affymetrix Y98 chips (GPL90) and raw image data was analyzed using the GeneChip Expression Analysis Software (Affymetrix). Probe set signals were processed by a Wilcoxon’s signed rank test and consolidated using a two-step linear mixed model. This sample is known as E3_T7_R3, 360 min. post rehydration.

organism: Saccharomyces cerevisiae


title: ECY_TCY_R2, Commercial dry active yeast

description:
Stock and seed cultures of strain BY4743 were grown in yeast extract-peptone-dextrose (YPD) medium (10 mg/ml yeast extract, 20 mg/ml bactopeptone, 20 mg/ml glucose, pH 6.5 ± 0.2), at 30°C until the A600 reached 0.8.  Cells were centrifuged and the cell pellet was resuspended and dispersed in 1:20 diluted YPD medium at between 22 to 25 °C.  An aliquot, which represent the control sample (T1), was removed for RNA extraction, and the remaining suspension was aliquoted into 9-cm diameter glass Petri dishes (15 ml/dish) for desiccation.
Desiccation was carried out in a custom-fabricated controlled atmosphere desiccation system, at 22 to 25 °C.  For more information on the unit and software, contact the corresponding author.  A series of aliquots were withdrawn from the cell suspensions 18h (T2) after the start of the drying process.
Samples were collected on a volume basis and were in the same ratio as collected at T1.  Once dry (42 h), the water potential was adjusted to 20% relative humidity for a period of 72 hr. A sample was collected at this timepoint, representing the dry sample (Tdry).  Dry cells were hydrated (22 to 25 °C ) using 1:20 diluted YPD medium, 15 ml per plate. Resuspended cells from the four plates were pooled in a sterile 250 ml flask.  Samples were collected for various analyses at time points of 15 min (T4), 45 min (T5), 90 min (T6) and 360 min (T7) after the start of rehydration.
Total RNA was isolated essentially as described (Schmitt, M. E., Brown, T. A., and Trumpower, B. L. (1990) Nucl. Acids Res. 18, 3091-3092).  RNA was quantified using a spectrophotometer, and its quality was assessed using the A260/A280 ratio.
Target cRNA was hybridized to Affymetrix Y98 chips (GPL90) and raw image data was analyzed using the GeneChip Expression Analysis Software (Affymetrix). Probe set signals were processed by a Wilcoxon’s signed rank test and consolidated using a two-step linear mixed model. This sample is known as ECY_TCY_R2, Commercial dry active yeast.

source_name: Commercial dry active yeast


title: ECY_TCY_R3, Commercial dry active yeast

description:
Stock and seed cultures of strain BY4743 were grown in yeast extract-peptone-dextrose (YPD) medium (10 mg/ml yeast extract, 20 mg/ml bactopeptone, 20 mg/ml glucose, pH 6.5 ± 0.2), at 30°C until the A600 reached 0.8.  Cells were centrifuged and the cell pellet was resuspended and dispersed in 1:20 diluted YPD medium at between 22 to 25 °C.  An aliquot, which represent the control sample (T1), was removed for RNA extraction, and the remaining suspension was aliquoted into 9-cm diameter glass Petri dishes (15 ml/dish) for desiccation.
Desiccation was carried out in a custom-fabricated controlled atmosphere desiccation system, at 22 to 25 °C.  For more information on the unit and software, contact the corresponding author.  A series of aliquots were withdrawn from the cell suspensions 18h (T2) after the start of the drying process.
Samples were collected on a volume basis and were in the same ratio as collected at T1.  Once dry (42 h), the water potential was adjusted to 20% relative humidity for a period of 72 hr. A sample was collected at this timepoint, representing the dry sample (Tdry).  Dry cells were hydrated (22 to 25 °C ) using 1:20 diluted YPD medium, 15 ml per plate. Resuspended cells from the four plates were pooled in a sterile 250 ml flask.  Samples were collected for various analyses at time points of 15 min (T4), 45 min (T5), 90 min (T6) and 360 min (T7) after the start of rehydration.
Total RNA was isolated essentially as described (Schmitt, M. E., Brown, T. A., and Trumpower, B. L. (1990) Nucl. Acids Res. 18, 3091-3092).  RNA was quantified using a spectrophotometer, and its quality was assessed using the A260/A280 ratio.
Target cRNA was hybridized to Affymetrix Y98 chips (GPL90) and raw image data was analyzed using the GeneChip Expression Analysis Software (Affymetrix). Probe set signals were processed by a Wilcoxon’s signed rank test and consolidated using a two-step linear mixed model. This sample is known as ECY_TCY_R3, Commercial dry active yeast.


title: E3_T5_R2, 45 min. post rehydration

description:
Stock and seed cultures of strain BY4743 were grown in yeast extract-peptone-dextrose (YPD) medium (10 mg/ml yeast extract, 20 mg/ml bactopeptone, 20 mg/ml glucose, pH 6.5 ± 0.2), at 30°C until the A600 reached 0.8.  Cells were centrifuged and the cell pellet was resuspended and dispersed in 1:20 diluted YPD medium at between 22 to 25 °C.  An aliquot, which represent the control sample (T1), was removed for RNA extraction, and the remaining suspension was aliquoted into 9-cm diameter glass Petri dishes (15 ml/dish) for desiccation.
Desiccation was carried out in a custom-fabricated controlled atmosphere desiccation system, at 22 to 25 °C.  For more information on the unit and software, contact the corresponding author.  A series of aliquots were withdrawn from the cell suspensions 18h (T2) after the start of the drying process.
Samples were collected on a volume basis and were in the same ratio as collected at T1.  Once dry (42 h), the water potential was adjusted to 20% relative humidity for a period of 72 hr. A sample was collected at this timepoint, representing the dry sample (Tdry).  Dry cells were hydrated (22 to 25 °C ) using 1:20 diluted YPD medium, 15 ml per plate. Resuspended cells from the four plates were pooled in a sterile 250 ml flask.  Samples were collected for various analyses at time points of 15 min (T4), 45 min (T5), 90 min (T6) and 360 min (T7) after the start of rehydration.
Total RNA was isolated essentially as described (Schmitt, M. E., Brown, T. A., and Trumpower, B. L. (1990) Nucl. Acids Res. 18, 3091-3092).  RNA was quantified using a spectrophotometer, and its quality was assessed using the A260/A280 ratio.
Target cRNA was hybridized to Affymetrix Y98 chips (GPL90) and raw image data was analyzed using the GeneChip Expression Analysis Software (Affymetrix). Probe set signals were processed by a Wilcoxon’s signed rank test and consolidated using a two-step linear mixed model. This sample is known as E1_T1_R1, Control.


title: Control 11, NC11

description:
Pathohistological and clinical examination of Patient NC11 (41 year-old female) yielded no significant pathological findings.  This patient was considered as a normal control for the purposes of the study.
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
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. ID (Location) 95A01 - 96P24 contained spotting buffer only and were flaged as empty / set to zero.
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: hMSCsVsLiver1

tag: Chemicon cat. Nr. 562


title: E3_T5_R1, 45 min. post rehydration


title: E3_T6_R2, 90 min. post rehydration

description:
Stock and seed cultures of strain BY4743 were grown in yeast extract-peptone-dextrose (YPD) medium (10 mg/ml yeast extract, 20 mg/ml bactopeptone, 20 mg/ml glucose, pH 6.5 ± 0.2), at 30°C until the A600 reached 0.8.  Cells were centrifuged and the cell pellet was resuspended and dispersed in 1:20 diluted YPD medium at between 22 to 25 °C.  An aliquot, which represent the control sample (T1), was removed for RNA extraction, and the remaining suspension was aliquoted into 9-cm diameter glass Petri dishes (15 ml/dish) for desiccation.
Desiccation was carried out in a custom-fabricated controlled atmosphere desiccation system, at 22 to 25 °C.  For more information on the unit and software, contact the corresponding author.  A series of aliquots were withdrawn from the cell suspensions 18h (T2) after the start of the drying process.
Samples were collected on a volume basis and were in the same ratio as collected at T1.  Once dry (42 h), the water potential was adjusted to 20% relative humidity for a period of 72 hr. A sample was collected at this timepoint, representing the dry sample (Tdry).  Dry cells were hydrated (22 to 25 °C ) using 1:20 diluted YPD medium, 15 ml per plate. Resuspended cells from the four plates were pooled in a sterile 250 ml flask.  Samples were collected for various analyses at time points of 15 min (T4), 45 min (T5), 90 min (T6) and 360 min (T7) after the start of rehydration.
Total RNA was isolated essentially as described (Schmitt, M. E., Brown, T. A., and Trumpower, B. L. (1990) Nucl. Acids Res. 18, 3091-3092).  RNA was quantified using a spectrophotometer, and its quality was assessed using the A260/A280 ratio.
Target cRNA was hybridized to Affymetrix Y98 chips (GPL90) and raw image data was analyzed using the GeneChip Expression Analysis Software (Affymetrix). Probe set signals were processed by a Wilcoxon’s signed rank test and consolidated using a two-step linear mixed model. This sample is known as E3_T6_R2, 90 min. post rehydration.


title: hMSCsVshMSC48h2


title: hMSCsVsBrain2

value: Foetal 6 weeks


title: hMSCsVshMSC6h2


title: ECY_TCY_R1, Commercial dry active yeast

description:
Stock and seed cultures of strain BY4743 were grown in yeast extract-peptone-dextrose (YPD) medium (10 mg/ml yeast extract, 20 mg/ml bactopeptone, 20 mg/ml glucose, pH 6.5 ± 0.2), at 30°C until the A600 reached 0.8.  Cells were centrifuged and the cell pellet was resuspended and dispersed in 1:20 diluted YPD medium at between 22 to 25 °C.  An aliquot, which represent the control sample (T1), was removed for RNA extraction, and the remaining suspension was aliquoted into 9-cm diameter glass Petri dishes (15 ml/dish) for desiccation.
Desiccation was carried out in a custom-fabricated controlled atmosphere desiccation system, at 22 to 25 °C.  For more information on the unit and software, contact the corresponding author.  A series of aliquots were withdrawn from the cell suspensions 18h (T2) after the start of the drying process.
Samples were collected on a volume basis and were in the same ratio as collected at T1.  Once dry (42 h), the water potential was adjusted to 20% relative humidity for a period of 72 hr. A sample was collected at this timepoint, representing the dry sample (Tdry).  Dry cells were hydrated (22 to 25 °C ) using 1:20 diluted YPD medium, 15 ml per plate. Resuspended cells from the four plates were pooled in a sterile 250 ml flask.  Samples were collected for various analyses at time points of 15 min (T4), 45 min (T5), 90 min (T6) and 360 min (T7) after the start of rehydration.
Total RNA was isolated essentially as described (Schmitt, M. E., Brown, T. A., and Trumpower, B. L. (1990) Nucl. Acids Res. 18, 3091-3092).  RNA was quantified using a spectrophotometer, and its quality was assessed using the A260/A280 ratio.
Target cRNA was hybridized to Affymetrix Y98 chips (GPL90) and raw image data was analyzed using the GeneChip Expression Analysis Software (Affymetrix). Probe set signals were processed by a Wilcoxon’s signed rank test and consolidated using a two-step linear mixed model. This sample is known as ECY_TCY_R1, Commercial dry active yeast.


title: E3_T6_R1, 90 min. post rehydration

description:
Stock and seed cultures of strain BY4743 were grown in yeast extract-peptone-dextrose (YPD) medium (10 mg/ml yeast extract, 20 mg/ml bactopeptone, 20 mg/ml glucose, pH 6.5 ± 0.2), at 30°C until the A600 reached 0.8.  Cells were centrifuged and the cell pellet was resuspended and dispersed in 1:20 diluted YPD medium at between 22 to 25 °C.  An aliquot, which represent the control sample (T1), was removed for RNA extraction, and the remaining suspension was aliquoted into 9-cm diameter glass Petri dishes (15 ml/dish) for desiccation.
Desiccation was carried out in a custom-fabricated controlled atmosphere desiccation system, at 22 to 25 °C.  For more information on the unit and software, contact the corresponding author.  A series of aliquots were withdrawn from the cell suspensions 18h (T2) after the start of the drying process.
Samples were collected on a volume basis and were in the same ratio as collected at T1.  Once dry (42 h), the water potential was adjusted to 20% relative humidity for a period of 72 hr. A sample was collected at this timepoint, representing the dry sample (Tdry).  Dry cells were hydrated (22 to 25 °C ) using 1:20 diluted YPD medium, 15 ml per plate. Resuspended cells from the four plates were pooled in a sterile 250 ml flask.  Samples were collected for various analyses at time points of 15 min (T4), 45 min (T5), 90 min (T6) and 360 min (T7) after the start of rehydration.
Total RNA was isolated essentially as described (Schmitt, M. E., Brown, T. A., and Trumpower, B. L. (1990) Nucl. Acids Res. 18, 3091-3092).  RNA was quantified using a spectrophotometer, and its quality was assessed using the A260/A280 ratio.
Target cRNA was hybridized to Affymetrix Y98 chips (GPL90) and raw image data was analyzed using the GeneChip Expression Analysis Software (Affymetrix). Probe set signals were processed by a Wilcoxon’s signed rank test and consolidated using a two-step linear mixed model. This sample is known as E3_T6_R1, 90 min. post rehydration.


title: E3_T6_R3, 90 min. post rehydration

description:
Stock and seed cultures of strain BY4743 were grown in yeast extract-peptone-dextrose (YPD) medium (10 mg/ml yeast extract, 20 mg/ml bactopeptone, 20 mg/ml glucose, pH 6.5 ± 0.2), at 30°C until the A600 reached 0.8.  Cells were centrifuged and the cell pellet was resuspended and dispersed in 1:20 diluted YPD medium at between 22 to 25 °C.  An aliquot, which represent the control sample (T1), was removed for RNA extraction, and the remaining suspension was aliquoted into 9-cm diameter glass Petri dishes (15 ml/dish) for desiccation.
Desiccation was carried out in a custom-fabricated controlled atmosphere desiccation system, at 22 to 25 °C.  For more information on the unit and software, contact the corresponding author.  A series of aliquots were withdrawn from the cell suspensions 18h (T2) after the start of the drying process.
Samples were collected on a volume basis and were in the same ratio as collected at T1.  Once dry (42 h), the water potential was adjusted to 20% relative humidity for a period of 72 hr. A sample was collected at this timepoint, representing the dry sample (Tdry).  Dry cells were hydrated (22 to 25 °C ) using 1:20 diluted YPD medium, 15 ml per plate. Resuspended cells from the four plates were pooled in a sterile 250 ml flask.  Samples were collected for various analyses at time points of 15 min (T4), 45 min (T5), 90 min (T6) and 360 min (T7) after the start of rehydration.
Total RNA was isolated essentially as described (Schmitt, M. E., Brown, T. A., and Trumpower, B. L. (1990) Nucl. Acids Res. 18, 3091-3092).  RNA was quantified using a spectrophotometer, and its quality was assessed using the A260/A280 ratio.
Target cRNA was hybridized to Affymetrix Y98 chips (GPL90) and raw image data was analyzed using the GeneChip Expression Analysis Software (Affymetrix). Probe set signals were processed by a Wilcoxon’s signed rank test and consolidated using a two-step linear mixed model. This sample is known as E3_T6_R3, 90 min. post rehydration.


title: E3_T4_R3, 15 min. post rehydration

description:
Stock and seed cultures of strain BY4743 were grown in yeast extract-peptone-dextrose (YPD) medium (10 mg/ml yeast extract, 20 mg/ml bactopeptone, 20 mg/ml glucose, pH 6.5 ± 0.2), at 30°C until the A600 reached 0.8.  Cells were centrifuged and the cell pellet was resuspended and dispersed in 1:20 diluted YPD medium at between 22 to 25 °C.  An aliquot, which represent the control sample (T1), was removed for RNA extraction, and the remaining suspension was aliquoted into 9-cm diameter glass Petri dishes (15 ml/dish) for desiccation.
Desiccation was carried out in a custom-fabricated controlled atmosphere desiccation system, at 22 to 25 °C.  For more information on the unit and software, contact the corresponding author.  A series of aliquots were withdrawn from the cell suspensions 18h (T2) after the start of the drying process.
Samples were collected on a volume basis and were in the same ratio as collected at T1.  Once dry (42 h), the water potential was adjusted to 20% relative humidity for a period of 72 hr. A sample was collected at this timepoint, representing the dry sample (Tdry).  Dry cells were hydrated (22 to 25 °C ) using 1:20 diluted YPD medium, 15 ml per plate. Resuspended cells from the four plates were pooled in a sterile 250 ml flask.  Samples were collected for various analyses at time points of 15 min (T4), 45 min (T5), 90 min (T6) and 360 min (T7) after the start of rehydration.
Total RNA was isolated essentially as described (Schmitt, M. E., Brown, T. A., and Trumpower, B. L. (1990) Nucl. Acids Res. 18, 3091-3092).  RNA was quantified using a spectrophotometer, and its quality was assessed using the A260/A280 ratio.
Target cRNA was hybridized to Affymetrix Y98 chips (GPL90) and raw image data was analyzed using the GeneChip Expression Analysis Software (Affymetrix). Probe set signals were processed by a Wilcoxon’s signed rank test and consolidated using a two-step linear mixed model. This sample is known as E3_T4_R3, 15 min. post rehydration.


title: E3_T5_R3, 45 min. post rehydration


title: hMSCsVshMSC48h1


title: E3_T7_R2, 360 min. post rehydration

description:
Stock and seed cultures of strain BY4743 were grown in yeast extract-peptone-dextrose (YPD) medium (10 mg/ml yeast extract, 20 mg/ml bactopeptone, 20 mg/ml glucose, pH 6.5 ± 0.2), at 30°C until the A600 reached 0.8.  Cells were centrifuged and the cell pellet was resuspended and dispersed in 1:20 diluted YPD medium at between 22 to 25 °C.  An aliquot, which represent the control sample (T1), was removed for RNA extraction, and the remaining suspension was aliquoted into 9-cm diameter glass Petri dishes (15 ml/dish) for desiccation.
Desiccation was carried out in a custom-fabricated controlled atmosphere desiccation system, at 22 to 25 °C.  For more information on the unit and software, contact the corresponding author.  A series of aliquots were withdrawn from the cell suspensions 18h (T2) after the start of the drying process.
Samples were collected on a volume basis and were in the same ratio as collected at T1.  Once dry (42 h), the water potential was adjusted to 20% relative humidity for a period of 72 hr. A sample was collected at this timepoint, representing the dry sample (Tdry).  Dry cells were hydrated (22 to 25 °C ) using 1:20 diluted YPD medium, 15 ml per plate. Resuspended cells from the four plates were pooled in a sterile 250 ml flask.  Samples were collected for various analyses at time points of 15 min (T4), 45 min (T5), 90 min (T6) and 360 min (T7) after the start of rehydration.
Total RNA was isolated essentially as described (Schmitt, M. E., Brown, T. A., and Trumpower, B. L. (1990) Nucl. Acids Res. 18, 3091-3092).  RNA was quantified using a spectrophotometer, and its quality was assessed using the A260/A280 ratio.
Target cRNA was hybridized to Affymetrix Y98 chips (GPL90) and raw image data was analyzed using the GeneChip Expression Analysis Software (Affymetrix). Probe set signals were processed by a Wilcoxon’s signed rank test and consolidated using a two-step linear mixed model. This sample is known as E3_T7_R2, 360 min. post rehydration.


title: E3_T7_R1, 360 min. post rehydration

description:
Stock and seed cultures of strain BY4743 were grown in yeast extract-peptone-dextrose (YPD) medium (10 mg/ml yeast extract, 20 mg/ml bactopeptone, 20 mg/ml glucose, pH 6.5 ± 0.2), at 30°C until the A600 reached 0.8.  Cells were centrifuged and the cell pellet was resuspended and dispersed in 1:20 diluted YPD medium at between 22 to 25 °C.  An aliquot, which represent the control sample (T1), was removed for RNA extraction, and the remaining suspension was aliquoted into 9-cm diameter glass Petri dishes (15 ml/dish) for desiccation.
Desiccation was carried out in a custom-fabricated controlled atmosphere desiccation system, at 22 to 25 °C.  For more information on the unit and software, contact the corresponding author.  A series of aliquots were withdrawn from the cell suspensions 18h (T2) after the start of the drying process.
Samples were collected on a volume basis and were in the same ratio as collected at T1.  Once dry (42 h), the water potential was adjusted to 20% relative humidity for a period of 72 hr. A sample was collected at this timepoint, representing the dry sample (Tdry).  Dry cells were hydrated (22 to 25 °C ) using 1:20 diluted YPD medium, 15 ml per plate. Resuspended cells from the four plates were pooled in a sterile 250 ml flask.  Samples were collected for various analyses at time points of 15 min (T4), 45 min (T5), 90 min (T6) and 360 min (T7) after the start of rehydration.
Total RNA was isolated essentially as described (Schmitt, M. E., Brown, T. A., and Trumpower, B. L. (1990) Nucl. Acids Res. 18, 3091-3092).  RNA was quantified using a spectrophotometer, and its quality was assessed using the A260/A280 ratio.
Target cRNA was hybridized to Affymetrix Y98 chips (GPL90) and raw image data was analyzed using the GeneChip Expression Analysis Software (Affymetrix). Probe set signals were processed by a Wilcoxon’s signed rank test and consolidated using a two-step linear mixed model. This sample is known as E3_T7_R1, 360 min. post rehydration.


title: E3_T4_R2, 15 min. post rehydration

description:
Stock and seed cultures of strain BY4743 were grown in yeast extract-peptone-dextrose (YPD) medium (10 mg/ml yeast extract, 20 mg/ml bactopeptone, 20 mg/ml glucose, pH 6.5 ± 0.2), at 30°C until the A600 reached 0.8.  Cells were centrifuged and the cell pellet was resuspended and dispersed in 1:20 diluted YPD medium at between 22 to 25 °C.  An aliquot, which represent the control sample (T1), was removed for RNA extraction, and the remaining suspension was aliquoted into 9-cm diameter glass Petri dishes (15 ml/dish) for desiccation.
Desiccation was carried out in a custom-fabricated controlled atmosphere desiccation system, at 22 to 25 °C.  For more information on the unit and software, contact the corresponding author.  A series of aliquots were withdrawn from the cell suspensions 18h (T2) after the start of the drying process.
Samples were collected on a volume basis and were in the same ratio as collected at T1.  Once dry (42 h), the water potential was adjusted to 20% relative humidity for a period of 72 hr. A sample was collected at this timepoint, representing the dry sample (Tdry).  Dry cells were hydrated (22 to 25 °C ) using 1:20 diluted YPD medium, 15 ml per plate. Resuspended cells from the four plates were pooled in a sterile 250 ml flask.  Samples were collected for various analyses at time points of 15 min (T4), 45 min (T5), 90 min (T6) and 360 min (T7) after the start of rehydration.
Total RNA was isolated essentially as described (Schmitt, M. E., Brown, T. A., and Trumpower, B. L. (1990) Nucl. Acids Res. 18, 3091-3092).  RNA was quantified using a spectrophotometer, and its quality was assessed using the A260/A280 ratio.
Target cRNA was hybridized to Affymetrix Y98 chips (GPL90) and raw image data was analyzed using the GeneChip Expression Analysis Software (Affymetrix). Probe set signals were processed by a Wilcoxon’s signed rank test and consolidated using a two-step linear mixed model. This sample is known as E3_T4_R2, 15 min. post rehydration.


title: E3_T3_R1, Dry

description:
Stock and seed cultures of strain BY4743 were grown in yeast extract-peptone-dextrose (YPD) medium (10 mg/ml yeast extract, 20 mg/ml bactopeptone, 20 mg/ml glucose, pH 6.5 ± 0.2), at 30°C until the A600 reached 0.8.  Cells were centrifuged and the cell pellet was resuspended and dispersed in 1:20 diluted YPD medium at between 22 to 25 °C.  An aliquot, which represent the control sample (T1), was removed for RNA extraction, and the remaining suspension was aliquoted into 9-cm diameter glass Petri dishes (15 ml/dish) for desiccation.
Desiccation was carried out in a custom-fabricated controlled atmosphere desiccation system, at 22 to 25 °C.  For more information on the unit and software, contact the corresponding author.  A series of aliquots were withdrawn from the cell suspensions 18h (T2) after the start of the drying process.
Samples were collected on a volume basis and were in the same ratio as collected at T1.  Once dry (42 h), the water potential was adjusted to 20% relative humidity for a period of 72 hr. A sample was collected at this timepoint, representing the dry sample (Tdry).  Dry cells were hydrated (22 to 25 °C ) using 1:20 diluted YPD medium, 15 ml per plate. Resuspended cells from the four plates were pooled in a sterile 250 ml flask.  Samples were collected for various analyses at time points of 15 min (T4), 45 min (T5), 90 min (T6) and 360 min (T7) after the start of rehydration.
Total RNA was isolated essentially as described (Schmitt, M. E., Brown, T. A., and Trumpower, B. L. (1990) Nucl. Acids Res. 18, 3091-3092).  RNA was quantified using a spectrophotometer, and its quality was assessed using the A260/A280 ratio.
Target cRNA was hybridized to Affymetrix Y98 chips (GPL90) and raw image data was analyzed using the GeneChip Expression Analysis Software (Affymetrix). Probe set signals were processed by a Wilcoxon’s signed rank test and consolidated using a two-step linear mixed model. This sample is known as E3_T3_R1, Dry.


title: E3_T2_R2, 50% dry

description:
Stock and seed cultures of strain BY4743 were grown in yeast extract-peptone-dextrose (YPD) medium (10 mg/ml yeast extract, 20 mg/ml bactopeptone, 20 mg/ml glucose, pH 6.5 ± 0.2), at 30°C until the A600 reached 0.8.  Cells were centrifuged and the cell pellet was resuspended and dispersed in 1:20 diluted YPD medium at between 22 to 25 °C.  An aliquot, which represent the control sample (T1), was removed for RNA extraction, and the remaining suspension was aliquoted into 9-cm diameter glass Petri dishes (15 ml/dish) for desiccation.
Desiccation was carried out in a custom-fabricated controlled atmosphere desiccation system, at 22 to 25 °C.  For more information on the unit and software, contact the corresponding author.  A series of aliquots were withdrawn from the cell suspensions 18h (T2) after the start of the drying process.
Samples were collected on a volume basis and were in the same ratio as collected at T1.  Once dry (42 h), the water potential was adjusted to 20% relative humidity for a period of 72 hr. A sample was collected at this timepoint, representing the dry sample (Tdry).  Dry cells were hydrated (22 to 25 °C ) using 1:20 diluted YPD medium, 15 ml per plate. Resuspended cells from the four plates were pooled in a sterile 250 ml flask.  Samples were collected for various analyses at time points of 15 min (T4), 45 min (T5), 90 min (T6) and 360 min (T7) after the start of rehydration.
Total RNA was isolated essentially as described (Schmitt, M. E., Brown, T. A., and Trumpower, B. L. (1990) Nucl. Acids Res. 18, 3091-3092).  RNA was quantified using a spectrophotometer, and its quality was assessed using the A260/A280 ratio.
Target cRNA was hybridized to Affymetrix Y98 chips (GPL90) and raw image data was analyzed using the GeneChip Expression Analysis Software (Affymetrix). Probe set signals were processed by a Wilcoxon’s signed rank test and consolidated using a two-step linear mixed model. This sample is known as E3_T2_R2, 50% dry.


title: E3_T1_R1, Control

description:
Stock and seed cultures of strain BY4743 were grown in yeast extract-peptone-dextrose (YPD) medium (10 mg/ml yeast extract, 20 mg/ml bactopeptone, 20 mg/ml glucose, pH 6.5 ± 0.2), at 30°C until the A600 reached 0.8.  Cells were centrifuged and the cell pellet was resuspended and dispersed in 1:20 diluted YPD medium at between 22 to 25 °C.  An aliquot, which represent the control sample (T1), was removed for RNA extraction, and the remaining suspension was aliquoted into 9-cm diameter glass Petri dishes (15 ml/dish) for desiccation.
Desiccation was carried out in a custom-fabricated controlled atmosphere desiccation system, at 22 to 25 °C.  For more information on the unit and software, contact the corresponding author.  A series of aliquots were withdrawn from the cell suspensions 18h (T2) after the start of the drying process.
Samples were collected on a volume basis and were in the same ratio as collected at T1.  Once dry (42 h), the water potential was adjusted to 20% relative humidity for a period of 72 hr. A sample was collected at this timepoint, representing the dry sample (Tdry).  Dry cells were hydrated (22 to 25 °C ) using 1:20 diluted YPD medium, 15 ml per plate. Resuspended cells from the four plates were pooled in a sterile 250 ml flask.  Samples were collected for various analyses at time points of 15 min (T4), 45 min (T5), 90 min (T6) and 360 min (T7) after the start of rehydration.
Total RNA was isolated essentially as described (Schmitt, M. E., Brown, T. A., and Trumpower, B. L. (1990) Nucl. Acids Res. 18, 3091-3092).  RNA was quantified using a spectrophotometer, and its quality was assessed using the A260/A280 ratio.
Target cRNA was hybridized to Affymetrix Y98 chips (GPL90) and raw image data was analyzed using the GeneChip Expression Analysis Software (Affymetrix). Probe set signals were processed by a Wilcoxon’s signed rank test and consolidated using a two-step linear mixed model. This sample is known as E3_T1_R1, Control.


title: E3_T3_R2, Dry

description:
Stock and seed cultures of strain BY4743 were grown in yeast extract-peptone-dextrose (YPD) medium (10 mg/ml yeast extract, 20 mg/ml bactopeptone, 20 mg/ml glucose, pH 6.5 ± 0.2), at 30°C until the A600 reached 0.8.  Cells were centrifuged and the cell pellet was resuspended and dispersed in 1:20 diluted YPD medium at between 22 to 25 °C.  An aliquot, which represent the control sample (T1), was removed for RNA extraction, and the remaining suspension was aliquoted into 9-cm diameter glass Petri dishes (15 ml/dish) for desiccation.
Desiccation was carried out in a custom-fabricated controlled atmosphere desiccation system, at 22 to 25 °C.  For more information on the unit and software, contact the corresponding author.  A series of aliquots were withdrawn from the cell suspensions 18h (T2) after the start of the drying process.
Samples were collected on a volume basis and were in the same ratio as collected at T1.  Once dry (42 h), the water potential was adjusted to 20% relative humidity for a period of 72 hr. A sample was collected at this timepoint, representing the dry sample (Tdry).  Dry cells were hydrated (22 to 25 °C ) using 1:20 diluted YPD medium, 15 ml per plate. Resuspended cells from the four plates were pooled in a sterile 250 ml flask.  Samples were collected for various analyses at time points of 15 min (T4), 45 min (T5), 90 min (T6) and 360 min (T7) after the start of rehydration.
Total RNA was isolated essentially as described (Schmitt, M. E., Brown, T. A., and Trumpower, B. L. (1990) Nucl. Acids Res. 18, 3091-3092).  RNA was quantified using a spectrophotometer, and its quality was assessed using the A260/A280 ratio.
Target cRNA was hybridized to Affymetrix Y98 chips (GPL90) and raw image data was analyzed using the GeneChip Expression Analysis Software (Affymetrix). Probe set signals were processed by a Wilcoxon’s signed rank test and consolidated using a two-step linear mixed model. This sample is known as E3_T3_R2, Dry.


title: E3_T2_R3, 50% dry

description:
Stock and seed cultures of strain BY4743 were grown in yeast extract-peptone-dextrose (YPD) medium (10 mg/ml yeast extract, 20 mg/ml bactopeptone, 20 mg/ml glucose, pH 6.5 ± 0.2), at 30°C until the A600 reached 0.8.  Cells were centrifuged and the cell pellet was resuspended and dispersed in 1:20 diluted YPD medium at between 22 to 25 °C.  An aliquot, which represent the control sample (T1), was removed for RNA extraction, and the remaining suspension was aliquoted into 9-cm diameter glass Petri dishes (15 ml/dish) for desiccation.
Desiccation was carried out in a custom-fabricated controlled atmosphere desiccation system, at 22 to 25 °C.  For more information on the unit and software, contact the corresponding author.  A series of aliquots were withdrawn from the cell suspensions 18h (T2) after the start of the drying process.
Samples were collected on a volume basis and were in the same ratio as collected at T1.  Once dry (42 h), the water potential was adjusted to 20% relative humidity for a period of 72 hr. A sample was collected at this timepoint, representing the dry sample (Tdry).  Dry cells were hydrated (22 to 25 °C ) using 1:20 diluted YPD medium, 15 ml per plate. Resuspended cells from the four plates were pooled in a sterile 250 ml flask.  Samples were collected for various analyses at time points of 15 min (T4), 45 min (T5), 90 min (T6) and 360 min (T7) after the start of rehydration.
Total RNA was isolated essentially as described (Schmitt, M. E., Brown, T. A., and Trumpower, B. L. (1990) Nucl. Acids Res. 18, 3091-3092).  RNA was quantified using a spectrophotometer, and its quality was assessed using the A260/A280 ratio.
Target cRNA was hybridized to Affymetrix Y98 chips (GPL90) and raw image data was analyzed using the GeneChip Expression Analysis Software (Affymetrix). Probe set signals were processed by a Wilcoxon’s signed rank test and consolidated using a two-step linear mixed model. This sample is known as E3_T2_R3, 50% dry.


title: E3_T2_R1, 50% dry

description:
Stock and seed cultures of strain BY4743 were grown in yeast extract-peptone-dextrose (YPD) medium (10 mg/ml yeast extract, 20 mg/ml bactopeptone, 20 mg/ml glucose, pH 6.5 ± 0.2), at 30°C until the A600 reached 0.8.  Cells were centrifuged and the cell pellet was resuspended and dispersed in 1:20 diluted YPD medium at between 22 to 25 °C.  An aliquot, which represent the control sample (T1), was removed for RNA extraction, and the remaining suspension was aliquoted into 9-cm diameter glass Petri dishes (15 ml/dish) for desiccation.
Desiccation was carried out in a custom-fabricated controlled atmosphere desiccation system, at 22 to 25 °C.  For more information on the unit and software, contact the corresponding author.  A series of aliquots were withdrawn from the cell suspensions 18h (T2) after the start of the drying process.
Samples were collected on a volume basis and were in the same ratio as collected at T1.  Once dry (42 h), the water potential was adjusted to 20% relative humidity for a period of 72 hr. A sample was collected at this timepoint, representing the dry sample (Tdry).  Dry cells were hydrated (22 to 25 °C ) using 1:20 diluted YPD medium, 15 ml per plate. Resuspended cells from the four plates were pooled in a sterile 250 ml flask.  Samples were collected for various analyses at time points of 15 min (T4), 45 min (T5), 90 min (T6) and 360 min (T7) after the start of rehydration.
Total RNA was isolated essentially as described (Schmitt, M. E., Brown, T. A., and Trumpower, B. L. (1990) Nucl. Acids Res. 18, 3091-3092).  RNA was quantified using a spectrophotometer, and its quality was assessed using the A260/A280 ratio.
Target cRNA was hybridized to Affymetrix Y98 chips (GPL90) and raw image data was analyzed using the GeneChip Expression Analysis Software (Affymetrix). Probe set signals were processed by a Wilcoxon’s signed rank test and consolidated using a two-step linear mixed model. This sample is known as E3_T2_R1, 50% dry.


title: E2_T7_R3, 360 min. post rehydration

description:
Stock and seed cultures of strain BY4743 were grown in yeast extract-peptone-dextrose (YPD) medium (10 mg/ml yeast extract, 20 mg/ml bactopeptone, 20 mg/ml glucose, pH 6.5 ± 0.2), at 30°C until the A600 reached 0.8.  Cells were centrifuged and the cell pellet was resuspended and dispersed in 1:20 diluted YPD medium at between 22 to 25 °C.  An aliquot, which represent the control sample (T1), was removed for RNA extraction, and the remaining suspension was aliquoted into 9-cm diameter glass Petri dishes (15 ml/dish) for desiccation.
Desiccation was carried out in a custom-fabricated controlled atmosphere desiccation system, at 22 to 25 °C.  For more information on the unit and software, contact the corresponding author.  A series of aliquots were withdrawn from the cell suspensions 18h (T2) after the start of the drying process.
Samples were collected on a volume basis and were in the same ratio as collected at T1.  Once dry (42 h), the water potential was adjusted to 20% relative humidity for a period of 72 hr. A sample was collected at this timepoint, representing the dry sample (Tdry).  Dry cells were hydrated (22 to 25 °C ) using 1:20 diluted YPD medium, 15 ml per plate. Resuspended cells from the four plates were pooled in a sterile 250 ml flask.  Samples were collected for various analyses at time points of 15 min (T4), 45 min (T5), 90 min (T6) and 360 min (T7) after the start of rehydration.
Total RNA was isolated essentially as described (Schmitt, M. E., Brown, T. A., and Trumpower, B. L. (1990) Nucl. Acids Res. 18, 3091-3092).  RNA was quantified using a spectrophotometer, and its quality was assessed using the A260/A280 ratio.
Target cRNA was hybridized to Affymetrix Y98 chips (GPL90) and raw image data was analyzed using the GeneChip Expression Analysis Software (Affymetrix). Probe set signals were processed by a Wilcoxon’s signed rank test and consolidated using a two-step linear mixed model. This sample is known as E2_T7_R3, 360 min. post rehydration.


title: E2_T6_R2, 90 min. post rehydration

description:
Stock and seed cultures of strain BY4743 were grown in yeast extract-peptone-dextrose (YPD) medium (10 mg/ml yeast extract, 20 mg/ml bactopeptone, 20 mg/ml glucose, pH 6.5 ± 0.2), at 30°C until the A600 reached 0.8.  Cells were centrifuged and the cell pellet was resuspended and dispersed in 1:20 diluted YPD medium at between 22 to 25 °C.  An aliquot, which represent the control sample (T1), was removed for RNA extraction, and the remaining suspension was aliquoted into 9-cm diameter glass Petri dishes (15 ml/dish) for desiccation.
Desiccation was carried out in a custom-fabricated controlled atmosphere desiccation system, at 22 to 25 °C.  For more information on the unit and software, contact the corresponding author.  A series of aliquots were withdrawn from the cell suspensions 18h (T2) after the start of the drying process.
Samples were collected on a volume basis and were in the same ratio as collected at T1.  Once dry (42 h), the water potential was adjusted to 20% relative humidity for a period of 72 hr. A sample was collected at this timepoint, representing the dry sample (Tdry).  Dry cells were hydrated (22 to 25 °C ) using 1:20 diluted YPD medium, 15 ml per plate. Resuspended cells from the four plates were pooled in a sterile 250 ml flask.  Samples were collected for various analyses at time points of 15 min (T4), 45 min (T5), 90 min (T6) and 360 min (T7) after the start of rehydration.
Total RNA was isolated essentially as described (Schmitt, M. E., Brown, T. A., and Trumpower, B. L. (1990) Nucl. Acids Res. 18, 3091-3092).  RNA was quantified using a spectrophotometer, and its quality was assessed using the A260/A280 ratio.
Target cRNA was hybridized to Affymetrix Y98 chips (GPL90) and raw image data was analyzed using the GeneChip Expression Analysis Software (Affymetrix). Probe set signals were processed by a Wilcoxon’s signed rank test and consolidated using a two-step linear mixed model. This sample is known as E2_T6_R2, 90 min. post rehydration.


title: E2_T6_R3, 90 min. post rehydration

description:
Stock and seed cultures of strain BY4743 were grown in yeast extract-peptone-dextrose (YPD) medium (10 mg/ml yeast extract, 20 mg/ml bactopeptone, 20 mg/ml glucose, pH 6.5 ± 0.2), at 30°C until the A600 reached 0.8.  Cells were centrifuged and the cell pellet was resuspended and dispersed in 1:20 diluted YPD medium at between 22 to 25 °C.  An aliquot, which represent the control sample (T1), was removed for RNA extraction, and the remaining suspension was aliquoted into 9-cm diameter glass Petri dishes (15 ml/dish) for desiccation.
Desiccation was carried out in a custom-fabricated controlled atmosphere desiccation system, at 22 to 25 °C.  For more information on the unit and software, contact the corresponding author.  A series of aliquots were withdrawn from the cell suspensions 18h (T2) after the start of the drying process.
Samples were collected on a volume basis and were in the same ratio as collected at T1.  Once dry (42 h), the water potential was adjusted to 20% relative humidity for a period of 72 hr. A sample was collected at this timepoint, representing the dry sample (Tdry).  Dry cells were hydrated (22 to 25 °C ) using 1:20 diluted YPD medium, 15 ml per plate. Resuspended cells from the four plates were pooled in a sterile 250 ml flask.  Samples were collected for various analyses at time points of 15 min (T4), 45 min (T5), 90 min (T6) and 360 min (T7) after the start of rehydration.
Total RNA was isolated essentially as described (Schmitt, M. E., Brown, T. A., and Trumpower, B. L. (1990) Nucl. Acids Res. 18, 3091-3092).  RNA was quantified using a spectrophotometer, and its quality was assessed using the A260/A280 ratio.
Target cRNA was hybridized to Affymetrix Y98 chips (GPL90) and raw image data was analyzed using the GeneChip Expression Analysis Software (Affymetrix). Probe set signals were processed by a Wilcoxon’s signed rank test and consolidated using a two-step linear mixed model. This sample is known as E2_T6_R3, 90 min. post rehydration.