title: wholeRefCy3 vs CNS1Cy5 610620GEO

description:
The MIAME Checklist
Experiment Design:
·	Type of experiment: for example, is it a comparison of normal vs. diseased tissue, a time course, or is it designed to study the effects of a gene knock-out?
Comparisons between individual tissues/organs at approximately 18 hours before pupariation staged according to Andres, A.J. & Thummel, C.S. Methods for quantitative analysis of transcription in larvae and prepupae. in Methods in Cell Biology, Volume 44.  Drosophila melanogaster: Practical Uses in Cell and Molecular Biology., Vol. 44 (eds. Goldstein, L.S.B. & Fyrberg, E.A.) 565-573 (Academic Press, San Diego, 1994)) vs. a reference sample from identically staged whole animal.  Abbreviations are: MG, midgut; SG, salivary gland; ED, epidermis with attached muscle; CNS, central nervous system; WD, wing disc.  RNA samples from Drosophila melanogaster (Canton S strain) were amplified according to Baugh, L. R., Hill, A. A., Brown, E. L., and Hunter, C. P. (2001). Quantitative analysis of mRNA amplification by in vitro transcription. Nucleic Acids Res 29, E29.  Samples from one tissue/organ were hybridized against the reference sample on each microarray.
·	Experimental factors: the parameters or conditions tested, such as time, dose, or genetic variation.
Each array tested differences in gene expression in different tissues/organs at one developmental stage.
·	The number of hybridizations performed in the experiment.
Three replicate hybridizations for each tissue using independent samples.
·	The type of reference used for the hybridizations, if any.
The reference sample is from identically staged whole animal.
·	Hybridization design: if applicable, a description of the comparisons made in each hybridization, whether to a standard reference sample, or between experimental samples. An accompanying diagram or table may be useful.
See above
·	Quality control steps taken: for example, replicates or dye swaps.
No dye swaps were used.  Three replicates for each tissue/organ.
·	URL of any supplemental websites or database accession numbers
Samples used, extract preparation and labeling:
·	The origin of the biological sample (for instance, name of the organism, the provider of the sample) and its characteristics: for example, gender, age, developmental stage, strain, or disease state.  See above.  Tissues/Organs were dissected from at least 12 individuals for each sample.
·	Manipulation of biological samples and protocols used: for example, growth conditions, treatments, separation techniques.  Animals were grown on standard corn meal medium with 0.05% bromophenol blue at 25 degrees Celsius.
·	Protocol for preparing the hybridization extract: for example, the RNA or DNA extraction and purification protocol
Total RNA was extracted using the SDS lysis protocol in Andres, A.J. & Thummel, C.S. “Methods for quantitative analysis of transcription in larvae and prepupae”. in  Methods in Cell Biology, Volume 44.  Drosophila melanogaster: Practical Uses in Cell and Molecular Biology., Vol. 44 (eds. Goldstein, L.S.B. & Fyrberg, E.A.) 565-573 (Academic Press, San Diego, 1994).  2 ug of total RNA from each sample was amplified with T7 RNA polymerase using a one-round linear amplification protocol according to Baugh, L. R., Hill, A. A., Brown, E. L., and Hunter, C. P. (2001). Quantitative analysis of mRNA amplification by in vitro transcription. Nucleic Acids Res 29, E29.
·	Labeling protocol(s).
Priming step with random hexamers.  Labeling step using Superscript II, buffer, DTT, dUTP-Cy3, dUTP-Cy5 and free nucleotides.  Cleanup using Qiagen PCR cleanup kit.  Hybridization to slide in a TE, SSC, SDS solution with poly-A as a blocker.  Hybridization chambers from DIE-TECH, incubated for 10 minutes at 42 degrees followed by approximately 15-hour incubation at 64 degrees.  Three washes with successively more dilute solutions of SSC, including SDS in the first wash, then a centrifuge spin to dry.  Arrays were stored in the dark until scanned.  See K.P. White and K.C. Burtis “Drosophila microarrays: from arrayer construction to hybridization” in Drosophila Protocols, Cold Spring Harbor Press, 2000 for microarray protocols.
·	External controls (spikes).
Hybridization procedures and parameters:
·	The protocol and conditions used during hybridization, blocking and washing.
See above.
Measurement data and specifications:
·	The quantitations based on the images.
Images were gridded and spots were quantified with GenePix software (Axon Instruments)
·	The set of quantitations from several arrays upon which the authors base their conclusions. While access to images of raw data is not required (although its value is unquestionable), authors should make every effort to provide the following:
o	Type of scanning hardware and software used: this information is appropriate for a materials and methods section.
Slides were scanned on an Axon 4000B scanner using Genepix Pro 3.0 software.
o	Type of image analysis software used: specifications should be stated in the materials and methods.
The program GenePix Pro 3.0 from Axon Instruments was used to analyse the data.
o	A description of the measurements produced by the image-analysis software and a description of which measurements were used in the analysis.
(F635 Median - B635), (F532 Median - B532), and (Ratio of Medians) were the raw measurements produced by the software.  The detailed information for these measurements is available at http://www.axon.com/manuals/GenePix_Pro_4.0_User_Guide_Rev_E.pdf
o	The complete output of the image analysis before data selection and transformation (spot quantitation matrices).
See *.xls files attached.
o	Data selection and transformation procedures.
Data for all spots are filtered.  For a spot to pass quality control, 3 conditions need to be met: (1). There is no automatic or manual flag during image gridding using GenePix Pro software.  (2). For this spot, at least 1 channel is of at least 75% of the pixels are one SD above background.  (3). The net signal noise ratio should be at least 1 for at least one channel.  For the channel 635 nm wavelength, the signal noise ratio is the ratio between (F635_median-B635_median) to the background median.  The background median is the bigger of the median of B635-median of all the spots on the whole array, and B-median of this spot.  For the channel 532 nm wavelength, F532_median and B532_median are used.  Ratios between net signals are log2 transformed and analyzed as detailed in the paper.
o	Final gene expression data table(s) used by the authors to make their conclusions after data selection and transformation (gene expression data matrices).
See the web tables attached for the paper.  Additionally, see the column for Passed and normalized ratio of median in the *.xls files attached.  Data before and after data selection is in one file.
Array Design:
·	General array design, including the platform type (whether the array is a spotted glass array, an in situ synthesized array, etc.); surface and coating specifications (when known – often commercial suppliers do not provide this data); and the availability of the array (the name or make of commercially available arrays).
Spotted array using PCR fragments of approximately 95% of the genes in the D. melanogaster BDGP genome.  Slides were coated with polylysine, printed using a GeneMachines OmniGrid printer with DNA suspended in 3X SSC, and postprocessed using the DCE protocol of Diehl, F., Grahlmann, S., Beier, M. & Hoheisel, J.D. “Manufacturing DNA microarrays of high spot homogeneity and reduced background signal.” Nucleic Acids Research 29, e38 (2001).
·	For each feature (spot) on the array, its location on the array and the ID of its respective reporter (molecule present on each spot) should be given.
The KP numbers identify these and can be tied to the data files.  The ID of the respective reporter is included in the Excel file Web Table PCR Primer and Amplicon Sequence.xls.
·	For each reporter,

source_name: Central nervous system at approximately 18 hours before pupariation

source_name: A reference sample from whole animal at approximately 18 hours before pupariation


title: MGRef Cy3 vs 18BPFb Cy5 680600GEO

status: Public on Jul 25 2003

type: RNA

city: New Haven

first: Tong-Ruei

last: Li

state: CT

address: 333 Cedar St. Rm#NSB386

department: Department of Genetics

country: USA

institute: Yale University School of Medicine

description:
The MIAME Checklist
Experiment Design:
· Type of experiment: for example, is it a comparison of normal vs. diseased tissue, a time course, or is it designed to study the effects of a gene knock-out?
Comparisons between the midgut at time points 18 hr. BPF, 4 hr. BPF, PF, 2 hr. APF, 3 hr. APF, 4 hr. APF, 5 hr. APF, 6 hr. APF, 8 hr. APF, 10 hr. APF and 12 hr. APF., vs. a reference sample. Reference sample is a mixture of the midgut samples at time points 18 hr. BPF, 4 hr. BPF, PF, 2 hr. APF, 3 hr. APF, 4 hr. APF, 5 hr. APF, 6 hr. APF, 8 hr. APF, and 12 hr. APF (BPF: before puparium formation; PF: puparium formation, also known as WPP (white prepupa); APF: after puparium formation).  Animal staging is according to Andres, A.J. & Thummel, C.S. Methods for quantitative analysis of transcription in larvae and prepupae. in Methods in Cell Biology, Volume 44. Drosophila melanogaster: Practical Uses in Cell and Molecular Biology., Vol. 44 (eds. Goldstein, L.S.B. & Fyrberg, E.A.) 565-573 (Academic Press, San Diego, 1994)). The reference sample is a mixture of the above-described samples. RNA samples from Drosophila melanogaster (Canton S strain) were amplified according to Baugh, L. R., Hill, A. A., Brown, E. L., and Hunter, C. P. (2001). Quantitative analysis of mRNA amplification by in vitro transcription. Nucleic Acids Res 29, E29. Samples from the midgut at one stage were hybridized against the reference sample on each microarray.
· Experimental factors: the parameters or conditions tested, such as time, dose, or genetic variation.
Each array tested differences in gene expression at different times in development in the midgut.
· The number of hybridizations performed in the experiment.
Three replicate hybridizations for each tissue using independent samples.
· The type of reference used for the hybridizations, if any.
The reference sample is a mixture of the samples from different stages.
· Hybridization design: if applicable, a description of the comparisons made in each hybridization, whether to a standard reference sample, or between experimental samples. An accompanying diagram or table may be useful.
See above.
· Quality control steps taken: for example, replicates or dye swaps.
No dye swaps were used. Three replicates for each stage.
· URL of any supplemental websites or database accession numbers
Samples used, extract preparation and labeling:
· The origin of the biological sample (for instance, name of the organism, the provider of the sample) and its characteristics: for example, gender, age, developmental stage, strain, or disease state. See above. Tissues/Organs were dissected from at least 12 individuals for each sample.
· Manipulation of biological samples and protocols used: for example, growth conditions, treatments, separation techniques. Animals were grown on standard corn meal medium with 0.05% bromophenol blue at 25 degrees Celsius.
· Protocol for preparing the hybridization extract: for example, the RNA or DNA extraction and purification protocol
Total RNA was extracted using TriZOL. 2 ug of total RNA from each sample was amplified with T7 RNA polymerase using a one-round linear amplification protocol. RNA extraction and amplification were according to Baugh, L. R., Hill, A. A., Brown, E. L., and Hunter, C. P. (2001). Quantitative analysis of mRNA amplification by in vitro transcription. Nucleic Acids Res 29, E29.
· Labeling protocol(s).
Priming step with random hexamers. Labeling step using Superscript II, buffer, DTT, dUTP-Cy3, dUTP-Cy5 and free nucleotides. Cleanup using Qiagen PCR cleanup kit. Hybridization to slide in a TE, SSC, SDS solution with poly-A as a blocker. Hybridization chambers from DIE-TECH, incubated for 10 minutes at 42 degrees followed by approximately 15-hour incubation at 64 degrees. Three washes with successively more dilute solutions of SSC, including SDS in the first wash, then a centrifuge spin to dry. Arrays were stored in the dark until scanned. See K.P. White and K.C. Burtis "Drosophila microarrays: from arrayer construction to hybridization" in Drosophila Protocols, Cold Spring Harbor Press, 2000 for microarray protocols.
· External controls (spikes).
Hybridization procedures and parameters:
· The protocol and conditions used during hybridization, blocking and washing.
See above.
Measurement data and specifications:
· The quantitations based on the images.
Images were gridded and spots were quantified with GenePix software (Axon Instruments)
· The set of quantitations from several arrays upon which the authors base their conclusions. While access to images of raw data is not required (although its value is unquestionable), authors should make every effort to provide the following:
o Type of scanning hardware and software used: this information is appropriate for a materials and methods section.
Slides were scanned on an Axon 4000B scanner using Genepix Pro 3.0 software.
o Type of image analysis software used: specifications should be stated in the materials and methods.
The program GenePix Pro 3.0 from Axon Instruments was used to analyse the data.
o A description of the measurements produced by the image-analysis software and a description of which measurements were used in the analysis.
(F635 Median - B635), (F532 Median - B532), and (Ratio of Medians) were the raw measurements produced by the software. The detailed information for these measurements is available at http://www.axon.com/manuals/GenePix_Pro_4.0_User_Guide_Rev_E.pdf
o The complete output of the image analysis before data selection and transformation (spot quantitation matrices).
See *.xls files attached.
o Data selection and transformation procedures.
Data for all spots are filtered. For a spot to pass quality control, 3 conditions need to be met: (1). There is no automatic or manual flag during image gridding using GenePix Pro software. (2). For this spot, at least 1 channel is of at least 75% of the pixels are one SD above background. (3). The net signal noise ratio should be at least 1 for at least one channel. For the channel at 635 nm wavelength, the signal noise ratio is the ratio between (F635_median-B635_median) and the background median. The background median is the bigger of the median of B635-median of all the spots on the whole array, and B-median of this spot. For the channel at 532 nm wavelength, F532_median and B532_median are used. Ratios between net signals are log2 transformed and analyzed as detailed in the paper.
o Final gene expression data table(s) used by the authors to make their conclusions after data selection and transformation (gene expression data matrices).
See the web tables attached for the paper. Additionally, see the column for Passed and normalized ratio of median in the *.xls files attached. Data before and after data selection is in one file.
Array Design:
· General array design, including the platform type (whether the array is a spotted glass array, an in situ synthesized array, etc.); surface and coating specifications (when known - often commercial suppliers do not provide this data); and the availability of the array (the name or make of commercially available arrays).
Spotted array using PCR fragments of approximately 95% of the genes in the D. melanogaster BDGP genome. Slides were coated with polylysine, printed using a GeneMachines OmniGrid printer with DNA suspended in 3X SSC, and postprocessed using the DCE protocol of Diehl, F., Grahlmann, S., Beier, M. & Hoheisel, J.D. "Manufacturing DNA microarrays of high spot homogeneity and reduced background signal." Nucleic Acids Research 29, e38 (2001).
· For each feature (spot) on the array, its location on the array and the ID of its respective reporter (molecule present on each spot) should be given.
The KP numbers identify these and can be tied to the data files. The ID of the respective reporter is included in the Excel file Web Table PCR Primer and Amplicon Sequence.xls.
· For each reporter, its type (e.g., cDNA or oligonucleotide) should be given, along with information that characterizes the reporter molecule unambiguously, in the form of appropriate database reference(s) and sequence (if available).
The FBGN and CG numbers, primer pairs, sequences, and KP number (to tie them to the spots on the array) are all listed in the Excel file Web Table PCR Primer and Amplicon Sequence.xls.
· For commercial arrays: a reference to the manufacturer should be provided, including a catalogue number and references to the manufacturer's website if available.
· For non-commercial arrays, the following details should be provided:
o The source of the reporter molecules: for example, the cDNA or oligo collection used, with references.
o The method of reporter preparation.
o The spotting protocols used, including the array substrate, the spotting buffer, and any post-printing processing, including cross-linking.
o Any additional treatment performed prior to hybridization.
We followed a sequence of decreasingly stringent tests to pick optimal primer pairs for the PCR fragments on the arrays. For each gene, we looked for (first) a 500b to 2kb sequence at the 3' end and, failing this, (second) two 200b to 500b sequences in:(1) coding sequence, completely within an EST clot, and unduplicated (BLAST<=10^-4); (2) coding sequence, partially within an EST clot, unduplicated; (3) coding sequence, unduplicated; (4) exon, completely within an EST clot, unduplicated; (5) exon, partially within an EST clot, unduplicated; (6) exon, unduplicated; (7) sequence less than 2kb at the 3' end. See above for the other details about making these arrays.
Sample hybridized to platform version GEO_Dmel20010730arrayplatform
Keywords = Drosophila, ecdysone, network, genomic, microarray, organogenesis, EcR, midgut, central nervous system, salivary gland, epidermis, imaginal disc, development

molecule: total RNA

organism: Drosophila melanogaster

source_name: 635nm (Cy5) channel, midgut at time point 18 hr. before puparium formation

source_name: 532nm (Cy3) channel, reference sample


title: wholeRefCy3 vs CNS2Cy5 700600GEO


title: MGRef Cy3 vs 18BPFa Cy5 670550GEO

description:
The MIAME Checklist
Experiment Design:
· Type of experiment: for example, is it a comparison of normal vs. diseased tissue, a time course, or is it designed to study the effects of a gene knock-out?
Comparisons between the midgut at time points 18 hr. BPF, 4 hr. BPF, PF, 2 hr. APF, 3 hr. APF, 4 hr. APF, 5 hr. APF, 6 hr. APF, 8 hr. APF, 10 hr. APF and 12 hr. APF., vs. a reference sample. Reference sample is a mixture of the midgut samples at time points 18 hr. BPF, 4 hr. BPF, PF, 2 hr. APF, 3 hr. APF, 4 hr. APF, 5 hr. APF, 6 hr. APF, 8 hr. APF, and 12 hr. APF (BPF: before puparium formation; PF: puparium formation, also known as WPP (white prepupa); APF: after puparium formation).  Animal staging is according to Andres, A.J. & Thummel, C.S. Methods for quantitative analysis of transcription in larvae and prepupae. in Methods in Cell Biology, Volume 44. Drosophila melanogaster: Practical Uses in Cell and Molecular Biology., Vol. 44 (eds. Goldstein, L.S.B. & Fyrberg, E.A.) 565-573 (Academic Press, San Diego, 1994)). The reference sample is a mixture of the above-described samples. RNA samples from Drosophila melanogaster (Canton S strain) were amplified according to Baugh, L. R., Hill, A. A., Brown, E. L., and Hunter, C. P. (2001). Quantitative analysis of mRNA amplification by in vitro transcription. Nucleic Acids Res 29, E29. Samples from the midgut at one stage were hybridized against the reference sample on each microarray.
· Experimental factors: the parameters or conditions tested, such as time, dose, or genetic variation.
Each array tested differences in gene expression at different times in development in the midgut.
· The number of hybridizations performed in the experiment.
Three replicate hybridizations for each tissue using independent samples.
· The type of reference used for the hybridizations, if any.
The reference sample is a mixture of the samples from different stages.
· Hybridization design: if applicable, a description of the comparisons made in each hybridization, whether to a standard reference sample, or between experimental samples. An accompanying diagram or table may be useful.
See above.
· Quality control steps taken: for example, replicates or dye swaps.
No dye swaps were used. Three replicates for each stage.
· URL of any supplemental websites or database accession numbers
Samples used, extract preparation and labeling:
· The origin of the biological sample (for instance, name of the organism, the provider of the sample) and its characteristics: for example, gender, age, developmental stage, strain, or disease state. See above. Tissues/Organs were dissected from at least 12 individuals for each sample.
· Manipulation of biological samples and protocols used: for example, growth conditions, treatments, separation techniques. Animals were grown on standard corn meal medium with 0.05% bromophenol blue at 25 degrees Celsius.
· Protocol for preparing the hybridization extract: for example, the RNA or DNA extraction and purification protocol
Total RNA was extracted using TriZOL. 2 ug of total RNA from each sample was amplified with T7 RNA polymerase using a one-round linear amplification protocol. RNA extraction and amplification were according to Baugh, L. R., Hill, A. A., Brown, E. L., and Hunter, C. P. (2001). Quantitative analysis of mRNA amplification by in vitro transcription. Nucleic Acids Res 29, E29.
· Labeling protocol(s).
Priming step with random hexamers. Labeling step using Superscript II, buffer, DTT, dUTP-Cy3, dUTP-Cy5 and free nucleotides. Cleanup using Qiagen PCR cleanup kit. Hybridization to slide in a TE, SSC, SDS solution with poly-A as a blocker. Hybridization chambers from DIE-TECH, incubated for 10 minutes at 42 degrees followed by approximately 15-hour incubation at 64 degrees. Three washes with successively more dilute solutions of SSC, including SDS in the first wash, then a centrifuge spin to dry. Arrays were stored in the dark until scanned. See K.P. White and K.C. Burtis "Drosophila microarrays: from arrayer construction to hybridization" in Drosophila Protocols, Cold Spring Harbor Press, 2000 for microarray protocols.
· External controls (spikes).
Hybridization procedures and parameters:
· The protocol and conditions used during hybridization, blocking and washing.
See above.
Measurement data and specifications:
· The quantitations based on the images.
Images were gridded and spots were quantified with GenePix software (Axon Instruments)
· The set of quantitations from several arrays upon which the authors base their conclusions. While access to images of raw data is not required (although its value is unquestionable), authors should make every effort to provide the following:
o Type of scanning hardware and software used: this information is appropriate for a materials and methods section.
Slides were scanned on an Axon 4000B scanner using Genepix Pro 3.0 software.
o Type of image analysis software used: specifications should be stated in the materials and methods.
The program GenePix Pro 3.0 from Axon Instruments was used to analyse the data.
o A description of the measurements produced by the image-analysis software and a description of which measurements were used in the analysis.
(F635 Median - B635), (F532 Median - B532), and (Ratio of Medians) were the raw measurements produced by the software. The detailed information for these measurements is available at http://www.axon.com/manuals/GenePix_Pro_4.0_User_Guide_Rev_E.pdf
o The complete output of the image analysis before data selection and transformation (spot quantitation matrices).
See *.xls files attached.
o Data selection and transformation procedures.
Data for all spots are filtered. For a spot to pass quality control, 3 conditions need to be met: (1). There is no automatic or manual flag during image gridding using GenePix Pro software. (2). For this spot, at least 1 channel is of at least 75% of the pixels are one SD above background. (3). The net signal noise ratio should be at least 1 for at least one channel. For the channel at 635 nm wavelength, the signal noise ratio is the ratio between (F635_median-B635_median) and the background median. The background median is the bigger of the median of B635-median of all the spots on the whole array, and B-median of this spot. For the channel at 532 nm wavelength, F532_median and B532_median are used. Ratios between net signals are log2 transformed and analyzed as detailed in the paper.
o Final gene expression data table(s) used by the authors to make their conclusions after data selection and transformation (gene expression data matrices).
See the web tables attached for the paper. Additionally, see the column for Passed and normalized ratio of median in the *.xls files attached. Data before and after data selection is in one file.
Array Design:
· General array design, including the platform type (whether the array is a spotted glass array, an in situ synthesized array, etc.); surface and coating specifications (when known - often commercial suppliers do not provide this data); and the availability of the array (the name or make of commercially available arrays).
Spotted array using PCR fragments of approximately 95% of the genes in the D. melanogaster BDGP genome. Slides were coated with polylysine, printed using a GeneMachines OmniGrid printer with DNA suspended in 3X SSC, and postprocessed using the DCE protocol of Diehl, F., Grahlmann, S., Beier, M. & Hoheisel, J.D. "Manufacturing DNA microarrays of high spot homogeneity and reduced background signal." Nucleic Acids Research 29, e38 (2001).
· For each feature (spot) on the array, its location on the array and the ID of its respective reporter (molecule present on each spot) should be given.
The KP numbers identify these and can be tied to the data files. The ID of the respective reporter is included in the Excel file Web Table PCR Primer and Amplicon Sequence.xls.
· For each reporter, its type (e.g., cDNA or oligonucleotide) should be given, along with information that characterizes the reporter molecule unambiguously, in the form of appropriate database reference(s) and sequence (if available).
The FBGN and CG numbers, primer pairs, sequences, and KP number (to tie them to the spots on the array) are all listed in the Excel file Web Table PCR Primer and Amplicon Sequence.xls.
· For commercial arrays: a reference to the manufacturer should be provided, including a catalogue number and references to the manufacturer's website if available.
· For non-commercial arrays, the following details should be provided:
o The source of the reporter molecules: for example, the cDNA or oligo collection used, with references.
o The method of reporter preparation.
o The spotting protocols used, including the array substrate, the spotting buffer, and any post-printing processing, including cross-linking.
o Any additional treatment performed prior to hybridization.
We followed a sequence of decreasingly stringent tests to pick optimal primer pairs for the PCR fragments on the arrays. For each gene, we looked for (first) a 500b to 2kb sequence at the 3' end and, failing this, (second) two 200b to 500b sequences in:(1) coding sequence, completely within an EST clot, and unduplicated (BLAST<=10^-4); (2) coding sequence, partially within an EST clot, unduplicated; (3) coding sequence, unduplicated; (4) exon, completely within an EST clot, unduplicated; (5) exon, partially within an EST clot, unduplicated; (6) exon, unduplicated; (7) sequence less than 2kb at the 3' end. See above for the other details about making these arrays.
Sample hybridized to platform version GEO_Dmel200102arrayplatform
Keywords = Drosophila, ecdysone, network, genomic, microarray, organogenesis, EcR, midgut, central nervous system, salivary gland, epidermis, imaginal disc, development


title: MGRef Cy3 vs 18BPFc Cy5 630600GEO


title: wholeRefCy3 vs CNS3Cy5 730600GEO


title: wholeRefCy3 vs ED2Cy5 700600GEO

description:
The MIAME Checklist
Experiment Design:
·	Type of experiment: for example, is it a comparison of normal vs. diseased tissue, a time course, or is it designed to study the effects of a gene knock-out?
Comparisons between individual tissues/organs at approximately 18 hours before pupariation staged according to Andres, A.J. & Thummel, C.S. Methods for quantitative analysis of transcription in larvae and prepupae. in Methods in Cell Biology, Volume 44.  Drosophila melanogaster: Practical Uses in Cell and Molecular Biology., Vol. 44 (eds. Goldstein, L.S.B. & Fyrberg, E.A.) 565-573 (Academic Press, San Diego, 1994)) vs. a reference sample from identically staged whole animal.  Abbreviations are: MG, Midgut; SG, salivary gland; ED, epidermis with attached muscle; CNS, central nervous system; WD, wing disc.  RNA samples from Drosophila melanogaster (Canton S strain) were amplified according to Baugh, L. R., Hill, A. A., Brown, E. L., and Hunter, C. P. (2001). Quantitative analysis of mRNA amplification by in vitro transcription. Nucleic Acids Res 29, E29.  Samples from one tissue/organ were hybridized against the reference sample on each microarray.
·	Experimental factors: the parameters or conditions tested, such as time, dose, or genetic variation.
Each array tested differences in gene expression in different tissues/organs at one developmental stage.
·	The number of hybridizations performed in the experiment.
Three replicate hybridizations for each tissue using independent samples.
·	The type of reference used for the hybridizations, if any.
The reference sample is from identically staged whole animal.
·	Hybridization design: if applicable, a description of the comparisons made in each hybridization, whether to a standard reference sample, or between experimental samples. An accompanying diagram or table may be useful.
See above
·	Quality control steps taken: for example, replicates or dye swaps.
No dye swaps were used.  Three replicates for each tissue/organ.
·	URL of any supplemental websites or database accession numbers
Samples used, extract preparation and labeling:
·	The origin of the biological sample (for instance, name of the organism, the provider of the sample) and its characteristics: for example, gender, age, developmental stage, strain, or disease state.  See above.  Tissues/Organs were dissected from at least 12 individuals for each sample.
·	Manipulation of biological samples and protocols used: for example, growth conditions, treatments, separation techniques.  Animals were grown on standard corn meal medium with 0.05% bromophenol blue at 25 degrees Celsius.
·	Protocol for preparing the hybridization extract: for example, the RNA or DNA extraction and purification protocol
Total RNA was extracted using the SDS lysis protocol in Andres, A.J. & Thummel, C.S. “Methods for quantitative analysis of transcription in larvae and prepupae”. in  Methods in Cell Biology, Volume 44.  Drosophila melanogaster: Practical Uses in Cell and Molecular Biology., Vol. 44 (eds. Goldstein, L.S.B. & Fyrberg, E.A.) 565-573 (Academic Press, San Diego, 1994).  2 ug of total RNA from each sample was amplified with T7 RNA polymerase using a one-round linear amplification protocol according to Baugh, L. R., Hill, A. A., Brown, E. L., and Hunter, C. P. (2001). Quantitative analysis of mRNA amplification by in vitro transcription. Nucleic Acids Res 29, E29.
·	Labeling protocol(s).
Priming step with random hexamers.  Labeling step using Superscript II, buffer, DTT, dUTP-Cy3, dUTP-Cy5 and free nucleotides.  Cleanup using Qiagen PCR cleanup kit.  Hybridization to slide in a TE, SSC, SDS solution with poly-A as a blocker.  Hybridization chambers from DIE-TECH, incubated for 10 minutes at 42 degrees followed by approximately 15-hour incubation at 64 degrees.  Three washes with successively more dilute solutions of SSC, including SDS in the first wash, then a centrifuge spin to dry.  Arrays were stored in the dark until scanned.  See K.P. White and K.C. Burtis “Drosophila microarrays: from arrayer construction to hybridization” in Drosophila Protocols, Cold Spring Harbor Press, 2000 for microarray protocols.
·	External controls (spikes).
Hybridization procedures and parameters:
·	The protocol and conditions used during hybridization, blocking and washing.
See above.
Measurement data and specifications:
·	The quantitations based on the images.
Images were gridded and spots were quantified with GenePix software (Axon Instruments)
·	The set of quantitations from several arrays upon which the authors base their conclusions. While access to images of raw data is not required (although its value is unquestionable), authors should make every effort to provide the following:
o	Type of scanning hardware and software used: this information is appropriate for a materials and methods section.
Slides were scanned on an Axon 4000B scanner using Genepix Pro 3.0 software.
o	Type of image analysis software used: specifications should be stated in the materials and methods.
The program GenePix Pro 3.0 from Axon Instruments was used to analyse the data.
o	A description of the measurements produced by the image-analysis software and a description of which measurements were used in the analysis.
(F635 Median - B635), (F532 Median - B532), and (Ratio of Medians) were the raw measurements produced by the software.  The detailed information for these measurements is available at http://www.axon.com/manuals/GenePix_Pro_4.0_User_Guide_Rev_E.pdf
o	The complete output of the image analysis before data selection and transformation (spot quantitation matrices).
See *.xls files attached.
o	Data selection and transformation procedures.
Data for all spots are filtered.  For a spot to pass quality control, 3 conditions need to be met: (1). There is no automatic or manual flag during image gridding using GenePix Pro software.  (2). For this spot, at least 1 channel is of at least 75% of the pixels are one SD above background.  (3). The net signal noise ratio should be at least 1 for at least one channel.  For the channel 635 nm wavelength, the signal noise ratio is the ratio between (F635_median-B635_median) to the background median.  The background median is the bigger of the median of B635-median of all the spots on the whole array, and B-median of this spot.  For the channel 532 nm wavelength, F532_median and B532_median are used.  Ratios between net signals are log2 transformed and analyzed as detailed in the paper.
o	Final gene expression data table(s) used by the authors to make their conclusions after data selection and transformation (gene expression data matrices).
See the web tables attached for the paper.  Additionally, see the column for Passed and normalized ratio of median in the *.xls files attached.  Data before and after data selection is in one file.
Array Design:
·	General array design, including the platform type (whether the array is a spotted glass array, an in situ synthesized array, etc.); surface and coating specifications (when known – often commercial suppliers do not provide this data); and the availability of the array (the name or make of commercially available arrays).
Spotted array using PCR fragments of approximately 95% of the genes in the D. melanogaster BDGP genome.  Slides were coated with polylysine, printed using a GeneMachines OmniGrid printer with DNA suspended in 3X SSC, and postprocessed using the DCE protocol of Diehl, F., Grahlmann, S., Beier, M. & Hoheisel, J.D. “Manufacturing DNA microarrays of high spot homogeneity and reduced background signal.” Nucleic Acids Research 29, e38 (2001).
·	For each feature (spot) on the array, its location on the array and the ID of its respective reporter (molecule present on each spot) should be given.
The KP numbers identify these and can be tied to the data files.  The ID of the respective reporter is included in the Excel file Web Table PCR Primer and Amplicon Sequence.xls.
·	For each reporter,

source_name: Epidermis with attached muscle at approximately 18 hours before pupariation


title: wholeRefCy3 vs ED3Cy5 620620GEO


title: wholeRefCy3 vs WD1Cy5 740600GEO

source_name: Wing disc at approximately 18 hours before pupariation


title: wholeRefCy3 vs WD3Cy5 800650GEO


title: wholeRefCy3 vs WD2Cy5 840640GEO


title: wholeRefCy3 vs MG3Cy5 710540GEO

source_name: Midgut at approximately 18 hours before pupariation


title: Bleo 129svJ 4 vs control 129svJ pool

status: Public on May 30 2003

city: San Francisco

first: Dean

last: Sheppard

state: CA

address: UCSF Box 0854

department: Medicine

institute: UCSF

description: Amplified RNA from lungs of bleomycin treated 129svJ 4 mouse vs. pool of amplified RNA from lungs of control 129svJ mice.

organism: Mus musculus

source_name: Bleo 129svJ 4

source_name: control 129svJ pool


title: wholeRefCy3 vs SG3Cy5 800500GEO

source_name: Salivary gland at approximately 18 hours before pupariation


title: Bleo A/J 4 vs control A/J pool

description: Amplified RNA from lungs of bleomycin treated A/J 4 mouse vs. pool of amplified RNA from lungs of control A/J mice.

source_name: Bleo A/J 4

source_name: control A/J pool


title: Bleo 129svJ 5vs control 129svJ pool

description: Amplified RNA from lungs of bleomycin treated 129svJ 5mouse vs. pool of amplified RNA from lungs of control 129svJ mice.

source_name: Bleo 129svJ 5


title: Bleo 129svJ 3 vs control 129svJ pool

description: Amplified RNA from lungs of bleomycin treated 129svJ 3 mouse vs. pool of amplified RNA from lungs of control 129svJ mice.

source_name: Bleo 129svJ 3


title: Bleo A/J 1 vs control A/J pool

description: Amplified RNA from lungs of bleomycin treated A/J 1 mouse vs. pool of amplified RNA from lungs of control A/J mice.

source_name: Bleo A/J 1


title: Bleo 129svJ 2 vs control 129svJ pool

description: Amplified RNA from lungs of bleomycin treated 129svJ 2 mouse vs. pool of amplified RNA from lungs of control 129svJ mice.

source_name: Bleo 129svJ 2


title: wholeRefCy3 vs MG2Cy5 630620GEO


title: Bleo A/J 3 vs control A/J pool

description: Amplified RNA from lungs of bleomycin treated A/J 3 mouse vs. pool of amplified RNA from lungs of control A/J mice.

source_name: Bleo A/J 3


title: EcRst2aMG3Cy5MGrefCy3_520450GEO

description:
The MIAME Checklist
Experiment Design:
·	Type of experiment: for example, is it a comparison of normal vs. diseased tissue, a time course, or is it designed to study the effects of a gene knock-out?
Comparisons between the midgut from EcR mutants vs. a reference sample.  EcR mutants (EcRM554fs/EcRV559fs) lacking the function of all EcR isoforms were collected at stage 2a, the arresting stage at the end of the third larval stage, using a heat shock inducible hs-EcR-B230.1 transgene as described (Li, T., and Bender, M. (2000). A conditional rescue system reveals essential functions for the ecdysone receptor (EcR) gene during molting and metamorphosis in Drosophila. Development 127, 2897-2905).  The reference sample is a mixture of the wild-type (Drosophila melanogaster (Canton S strain)) midgut samples at various stages, which is the same reference sample used for the wild-type midgut timecourse that is described in the paper.  Total RNA samples were amplified according to Baugh, L. R., Hill, A. A., Brown, E. L., and Hunter, C. P. (2001). Quantitative analysis of mRNA amplification by in vitro transcription. Nucleic Acids Res 29, E29.  Samples from the midgut of EcR mutants at one stage were hybridized against the reference sample on each microarray.
·	Experimental factors: the parameters or conditions tested, such as time, dose, or genetic variation.
Each array tested the gene expression in the midgut of EcR mutants.
·	The number of hybridizations performed in the experiment.
Three replicate hybridizations using independent samples.
·	The type of reference used for the hybridizations, if any.
The reference sample is a mixture of the wild-type samples from different stages.
·	Hybridization design: if applicable, a description of the comparisons made in each hybridization, whether to a standard reference sample, or between experimental samples. An accompanying diagram or table may be useful.
See above.
·	Quality control steps taken: for example, replicates or dye swaps.
No dye swaps were used.  Three replicates in total.
·	URL of any supplemental websites or database accession numbers
Samples used, extract preparation and labeling:
·	The origin of the biological sample (for instance, name of the organism, the provider of the sample) and its characteristics: for example, gender, age, developmental stage, strain, or disease state.  See above.  Tissues/Organs were dissected from at least 12 individuals for each sample.
·	Manipulation of biological samples and protocols used: for example, growth conditions, treatments, separation techniques.  Animals were grown on standard corn meal medium with 0.05% bromophenol blue at 25 degrees Celsius.
·	Protocol for preparing the hybridization extract: for example, the RNA or DNA extraction and purification protocol
Total RNA was extracted using TriZOL.  2 ug of total RNA from each sample was amplified with T7 RNA polymerase using a one-round linear amplification protocol.  RNA extraction and amplification were according to Baugh, L. R., Hill, A. A., Brown, E. L., and Hunter, C. P. (2001). Quantitative analysis of mRNA amplification by in vitro transcription. Nucleic Acids Res 29, E29.
·	Labeling protocol(s).
Priming step with random hexamers.  Labeling step using Superscript II, buffer, DTT, dUTP-Cy3, dUTP-Cy5 and free nucleotides.  Cleanup using Qiagen PCR cleanup kit.  Hybridization to slide in a TE, SSC, SDS solution with poly-A as a blocker.  Hybridization chambers from DIE-TECH, incubated for 10 minutes at 42 degrees followed by approximately 15-hour incubation at 64 degrees.  Three washes with successively more dilute solutions of SSC, including SDS in the first wash, then a centrifuge spin to dry.  Arrays were stored in the dark until scanned.  See K.P. White and K.C. Burtis “Drosophila microarrays: from arrayer construction to hybridization” in Drosophila Protocols, Cold Spring Harbor Press, 2000 for microarray protocols.
·	External controls (spikes).
Hybridization procedures and parameters:
·	The protocol and conditions used during hybridization, blocking and washing.
See above.
Measurement data and specifications:
·	The quantitations based on the images.
Images were gridded and spots were quantified with GenePix software (Axon Instruments)
·	The set of quantitations from several arrays upon which the authors base their conclusions. While access to images of raw data is not required (although its value is unquestionable), authors should make every effort to provide the following:
o	Type of scanning hardware and software used: this information is appropriate for a materials and methods section.
Slides were scanned on an Axon 4000B scanner using Genepix Pro 3.0 software.
o	Type of image analysis software used: specifications should be stated in the materials and methods.
The program GenePix Pro 3.0 from Axon Instruments was used to analyse the data.
o	A description of the measurements produced by the image-analysis software and a description of which measurements were used in the analysis.
(F635 Median - B635), (F532 Median - B532), and (Ratio of Medians) were the raw measurements produced by the software.  The detailed information for these measurements is available at http://www.axon.com/manuals/GenePix_Pro_4.0_User_Guide_Rev_E.pdf
o	The complete output of the image analysis before data selection and transformation (spot quantitation matrices).
See *.xls files attached.
o	Data selection and transformation procedures.
Data for all spots are filtered.  For a spot to pass quality control, 3 conditions need to be met: (1). There is no automatic or manual flag during image gridding using GenePix Pro software.  2. For this spot, at least 1 channel is of at least 75% of the pixels are one SD above background.  3. The net signal noise ratio should be at least 1 for at least one channel.  For the channel 635 nm wavelength, the signal noise ratio is the ratio between (F635_median-B635_median) to the background median.  The background median is the bigger of the median of B635-median of all the spots on the whole array, and B-median of this spot.  For the channel 532 nm wavelength, F532_median and B532_median are used.  Ratios between net signals are log2 transformed and analyzed as detailed in the paper.
o	Final gene expression data table(s) used by the authors to make their conclusions after data selection and transformation (gene expression data matrices).
See the web tables attached for the paper.  Additionally, see the column for Passed and normalized ratio of median in the *.xls files attached.  Data before and after data selection is in one file.
Array Design:
·	General array design, including the platform type (whether the array is a spotted glass array, an in situ synthesized array, etc.); surface and coating specifications (when known – often commercial suppliers do not provide this data); and the availability of the array (the name or make of commercially available arrays).
Spotted array using PCR fragments of approximately 95% of the genes in the D. melanogaster BDGP genome.  Slides were coated with polylysine, printed using a GeneMachines OmniGrid printer with DNA suspended in 3X SSC, and postprocessed using the DCE protocol of Diehl, F., Grahlmann, S., Beier, M. & Hoheisel, J.D. “Manufacturing DNA microarrays of high spot homogeneity and reduced background signal.” Nucleic Acids Research 29, e38 (2001).
·	For each feature (spot) on the array, its location on the array and the ID of its respective reporter (molecule present on each spot) should be given.
The KP numbers identify these and can be tied to the data files.  The ID of the respective reporter is included in the Excel file Web Table PCR Primer and Amplicon Sequence.xls.
·	For each reporter, its type (e.g., cDNA or oligonucleotide) should be given, along with information that characterizes the reporter molecule unambiguously, in the form of appropriate database reference(s) and sequence (if available).
The FBGN and CG n

source_name: 635nm Ch1, the midgut from EcR mutants vs. a reference sample.  EcR mutants (EcRM554fs/EcRV559fs) lacking the function of all EcR isoforms were collected at stage 2a

source_name: 532nm Ch2, the reference sample - wild type (Canton S) midgut samples at various stages


title: wholeRefCy3 vs SG2Cy5 710530GEO

source_name: A reference sample from whole animal at approximately 18 hours before pupariation


title: Ova A/J 2 vs control A/J pool

status: Public on May 29 2003

description: Amplified RNA from lungs of ovalbumen sensitized and challenged A/J 2 mouse vs. pool of amplified RNA from lungs of control A/J mice.

source_name: Ova A/J 2


title: wholeRefCy3 vs SG1Cy5 710520GEO


title: wholeRefCy3 vs ED1Cy5 740600GEO

source_name: Epidermis with attached muscle at approximately 18 hours before pupariation


title: Ova A/J 4 vs control A/J pool

description: Amplified RNA from lungs of ovalbumen sensitized and challenged A/J 4 mouse vs. pool of amplified RNA from lungs of control A/J mice.

source_name: Ova A/J 4


title: Ova A/J 6 vs control A/J pool

description: Amplified RNA from lungs of ovalbumen sensitized and challenged A/J 6 mouse vs. pool of amplified RNA from lungs of control A/J mice.

source_name: Ova A/J 6


title: Bleo A/J 2 vs control A/J pool

description: Amplified RNA from lungs of Bleomycin treated A/J 2 mouse vs. pool of amplified RNA from lungs of control A/J mice.

source_name: Bleo A/J 2


title: Ova A/J 1 vs control A/J pool

description: Amplified RNA from lungs of ovalbumen sensitized and challenged A/J 1 mouse vs. pool of amplified RNA from lungs of control A/J mice.

source_name: Ova A/J 1


title: Bleo 129svJ 1 vs control 129svJ pool

description: Amplified RNA from lungs of bleomycin treated 129svJ 1 mouse vs. pool of amplified RNA from lungs of control 129svJ mice.

source_name: Bleo 129svJ 1

source_name: control 129svJ pool


title: Ova A/J 5 vs control A/J pool

description: Amplified RNA from lungs of ovalbumen sensitized and challenged A/J 5 mouse vs. pool of amplified RNA from lungs of control A/J mice.

source_name: Ova A/J 5

source_name: control A/J pool


title: Ova A/J 3 vs control A/J pool

description: Amplified RNA from lungs of ovalbumen sensitized and challenged A/J 3 mouse vs. pool of amplified RNA from lungs of control A/J mice.

source_name: Ova A/J 3


title: Ova C57 6 vs control C57 pool

description: Amplified RNA from lungs of ovalbumen sensitized and challenged C57 6 mouse vs. pool of amplified RNA from lungs of control C57 mice.

source_name: Ova C57 6

source_name: control C57 pool


title: EcRst2aMG6Cy5MGrefCy3_530440GEO


title: wholeRefCy3 vs MG1Cy5 640620GEO

description:
The MIAME Checklist
Experiment Design:
·	Type of experiment: for example, is it a comparison of normal vs. diseased tissue, a time course, or is it designed to study the effects of a gene knock-out?
Comparisons between individual tissues/organs at approximately 18 hours before pupariation staged according to Andres, A.J. & Thummel, C.S. Methods for quantitative analysis of transcription in larvae and prepupae. in Methods in Cell Biology, Volume 44.  Drosophila melanogaster: Practical Uses in Cell and Molecular Biology., Vol. 44 (eds. Goldstein, L.S.B. & Fyrberg, E.A.) 565-573 (Academic Press, San Diego, 1994)) vs. a reference sample from identically staged whole animal.  Abbreviations are: MG, midgut; SG, salivary gland; ED, epidermis with attached muscle; CNS, central nervous system; WD, wing disc.  RNA samples from Drosophila melanogaster (Canton S strain) were amplified according to Baugh, L. R., Hill, A. A., Brown, E. L., and Hunter, C. P. (2001). Quantitative analysis of mRNA amplification by in vitro transcription. Nucleic Acids Res 29, E29.  Samples from one tissue/organ were hybridized against the reference sample on each microarray.
·	Experimental factors: the parameters or conditions tested, such as time, dose, or genetic variation.
Each array tested differences in gene expression in different tissues/organs at one developmental stage.
·	The number of hybridizations performed in the experiment.
Three replicate hybridizations for each tissue using independent samples.
·	The type of reference used for the hybridizations, if any.
The reference sample is from identically staged whole animal.
·	Hybridization design: if applicable, a description of the comparisons made in each hybridization, whether to a standard reference sample, or between experimental samples. An accompanying diagram or table may be useful.
See above
·	Quality control steps taken: for example, replicates or dye swaps.
No dye swaps were used.  Three replicates for each tissue/organ.
·	URL of any supplemental websites or database accession numbers
Samples used, extract preparation and labeling:
·	The origin of the biological sample (for instance, name of the organism, the provider of the sample) and its characteristics: for example, gender, age, developmental stage, strain, or disease state.  See above.  Tissues/Organs were dissected from at least 12 individuals for each sample.
·	Manipulation of biological samples and protocols used: for example, growth conditions, treatments, separation techniques.  Animals were grown on standard corn meal medium with 0.05% bromophenol blue at 25 degrees Celsius.
·	Protocol for preparing the hybridization extract: for example, the RNA or DNA extraction and purification protocol
Total RNA was extracted using the SDS lysis protocol in Andres, A.J. & Thummel, C.S. “Methods for quantitative analysis of transcription in larvae and prepupae”. in  Methods in Cell Biology, Volume 44.  Drosophila melanogaster: Practical Uses in Cell and Molecular Biology., Vol. 44 (eds. Goldstein, L.S.B. & Fyrberg, E.A.) 565-573 (Academic Press, San Diego, 1994).  2 ug of total RNA from each sample was amplified with T7 RNA polymerase using a one-round linear amplification protocol according to Baugh, L. R., Hill, A. A., Brown, E. L., and Hunter, C. P. (2001). Quantitative analysis of mRNA amplification by in vitro transcription. Nucleic Acids Res 29, E29.
·	Labeling protocol(s).
Priming step with random hexamers.  Labeling step using Superscript II, buffer, DTT, dUTP-Cy3, dUTP-Cy5 and free nucleotides.  Cleanup using Qiagen PCR cleanup kit.  Hybridization to slide in a TE, SSC, SDS solution with poly-A as a blocker.  Hybridization chambers from DIE-TECH, incubated for 10 minutes at 42 degrees followed by approximately 15-hour incubation at 64 degrees.  Three washes with successively more dilute solutions of SSC, including SDS in the first wash, then a centrifuge spin to dry.  Arrays were stored in the dark until scanned.  See K.P. White and K.C. Burtis “Drosophila microarrays: from arrayer construction to hybridization” in Drosophila Protocols, Cold Spring Harbor Press, 2000 for microarray protocols.
·	External controls (spikes).
Hybridization procedures and parameters:
·	The protocol and conditions used during hybridization, blocking and washing.
See above.
Measurement data and specifications:
·	The quantitations based on the images.
Images were gridded and spots were quantified with GenePix software (Axon Instruments)
·	The set of quantitations from several arrays upon which the authors base their conclusions. While access to images of raw data is not required (although its value is unquestionable), authors should make every effort to provide the following:
o	Type of scanning hardware and software used: this information is appropriate for a materials and methods section.
Slides were scanned on an Axon 4000B scanner using Genepix Pro 3.0 software.
o	Type of image analysis software used: specifications should be stated in the materials and methods.
The program GenePix Pro 3.0 from Axon Instruments was used to analyse the data.
o	A description of the measurements produced by the image-analysis software and a description of which measurements were used in the analysis.
(F635 Median - B635), (F532 Median - B532), and (Ratio of Medians) were the raw measurements produced by the software.  The detailed information for these measurements is available at http://www.axon.com/manuals/GenePix_Pro_4.0_User_Guide_Rev_E.pdf
o	The complete output of the image analysis before data selection and transformation (spot quantitation matrices).
See *.xls files attached.
o	Data selection and transformation procedures.
Data for all spots are filtered.  For a spot to pass quality control, 3 conditions need to be met: (1). There is no automatic or manual flag during image gridding using GenePix Pro software.  (2). For this spot, at least 1 channel is of at least 75% of the pixels are one SD above background.  (3). The net signal noise ratio should be at least 1 for at least one channel.  For the channel 635 nm wavelength, the signal noise ratio is the ratio between (F635_median-B635_median) to the background median.  The background median is the bigger of the median of B635-median of all the spots on the whole array, and B-median of this spot.  For the channel 532 nm wavelength, F532_median and B532_median are used.  Ratios between net signals are log2 transformed and analyzed as detailed in the paper.
o	Final gene expression data table(s) used by the authors to make their conclusions after data selection and transformation (gene expression data matrices).
See the web tables attached for the paper.  Additionally, see the column for Passed and normalized ratio of median in the *.xls files attached.  Data before and after data selection is in one file.
Array Design:
·	General array design, including the platform type (whether the array is a spotted glass array, an in situ synthesized array, etc.); surface and coating specifications (when known – often commercial suppliers do not provide this data); and the availability of the array (the name or make of commercially available arrays).
Spotted array using PCR fragments of approximately 95% of the genes in the D. melanogaster BDGP genome.  Slides were coated with polylysine, printed using a GeneMachines OmniGrid printer with DNA suspended in 3X SSC, and postprocessed using the DCE protocol of Diehl, F., Grahlmann, S., Beier, M. & Hoheisel, J.D. “Manufacturing DNA microarrays of high spot homogeneity and reduced background signal.” Nucleic Acids Research 29, e38 (2001).
·	For each feature (spot) on the array, its location on the array and the ID of its respective reporter (molecule present on each spot) should be given.
The KP numbers identify these and can be tied to the data files.  The ID of the respective reporter is included in the Excel file Web Table PCR Primer and Amplicon Sequence.xls.
·	For each reporter,

source_name: Midgut at approximately 18 hours before pupariation


title: Ova C57 3 vs control C57 pool

description: Amplified RNA from lungs of ovalbumen sensitized and challenged C57 3 mouse vs. pool of amplified RNA from lungs of control C57 mice.

source_name: Ova C57 3


title: Ova C57 1 vs control C57 pool

description: Amplified RNA from lungs of ovalbumen sensitized and challenged C57 1 mouse vs. pool of amplified RNA from lungs of control C57 mice.

source_name: Ova C57 1


title: Ova C57 2 vs control C57 pool

description: Amplified RNA from lungs of ovalbumen sensitized and challenged C57 2 mouse vs. pool of amplified RNA from lungs of control C57 mice.

source_name: Ova C57 2


title: Ova C57 4 vs control C57 pool

description: Amplified RNA from lungs of ovalbumen sensitized and challenged C57 4 mouse vs. pool of amplified RNA from lungs of control C57 mice.

source_name: Ova C57 4


title: Ova C3H 5 vs control C3H pool

description: Amplified RNA from lungs of ovalbumen sensitized and challenged C3H 5 mouse vs. pool of amplified RNA from lungs of control C3H mice.

source_name: Ova C3H 5

source_name: control C3H pool


title: SL2_vs_Kc167_S1_flip_1

status: Public on May 27 2003

city: Mississauga

first: Zak

last: Razak

state: ON

address: 3359 Mississauga Road

department: Zoology Department

country: Canada

institute: University of Toronto

description:
The expression profiles of Drosophila Kc167 and SL2 cell lines were compared.  Total RNA was extracted from dense cultures of Kc167 or SL2 cells which were maintained at 22C using Trizol Reagent (Invitrogen, Burlington, ON).   Kc167 cells were grown in Schneider's Complete Drosophila Medium (Invitrogen) supplemented with 5% Fetal Bovine Serum (Invitrogen) and 20ug/mL Gentamicin (Sigma, Oakville, ON) whereas SL2 cells were grown in CCM3 media (HyClone, Logan, UT) supplemented with 20ug/mL Gentamicin.  80ug of RNA, quantified by A260 in an Ultrmark plate reader (Biorad, Mississauga, ON), was used in each direct labeling reaction (see http://www.flyarrays.com) to generate fluorescently labeled cDNA.  The respective Cy3 (KC167) and Cy5 (SL2) targets were suspended in 75 uL DIG EasyHyb solution (Roche, Mississauga, ON)and were competitively hybridized to the array for 16 hours in a CMT-GAPS hybridization chamber (Corning, Acton, MA).  Scanning was performed with a ScanArray 4000 system (Perkin Elmer, Boston, MA) using the following settings:  10uM scan:  Cy3(543 nm) 100-72, Cy5(633 nm) 100-66 (laser power-PMT Gain).  Quantification was performed using QuantArray v3 software (Perkin Elmer) using the adaptive algorithm without background subtraction.  The data in the file is not normalized.  The ratio in the Value column was derived from normalized data (not shown) as discussed in the manuscript.
Keywords = Kc167
Keywords = SL2
Keywords = Drosophila
Lot batch = d7k2-070802

source_name: Untreated Kc167 cells

source_name: Untreated SL2 cells


title: SL2_vs_Kc167_S3_flip_1


title: EcRst2aMG4Cy5MGrefCy3_530450GEO

description:
The MIAME Checklist
Experiment Design:
·	Type of experiment: for example, is it a comparison of normal vs. diseased tissue, a time course, or is it designed to study the effects of a gene knock-out?
Comparisons between the midgut from EcR mutants vs. a reference sample.  EcR mutants (EcRM554fs/EcRV559fs) lacking the function of all EcR isoforms were collected at stage 2a, the arresting stage at the end of the third larval stage, using a heat shock inducible hs-EcR-B230.1 transgene as described (Li, T., and Bender, M. (2000). A conditional rescue system reveals essential functions for the ecdysone receptor (EcR) gene during molting and metamorphosis in Drosophila. Development 127, 2897-2905).  The reference sample is a mixture of the wild-type (Drosophila melanogaster (Canton S strain)) midgut samples at various stages, which is the same reference sample used for the wild-type midgut timecourse that is described in the paper.  Total RNA samples were amplified according to Baugh, L. R., Hill, A. A., Brown, E. L., and Hunter, C. P. (2001). Quantitative analysis of mRNA amplification by in vitro transcription. Nucleic Acids Res 29, E29.  Samples from the midgut of EcR mutants at one stage were hybridized against the reference sample on each microarray.
·	Experimental factors: the parameters or conditions tested, such as time, dose, or genetic variation.
Each array tested the gene expression in the midgut of EcR mutants.
·	The number of hybridizations performed in the experiment.
Three replicate hybridizations using independent samples.
·	The type of reference used for the hybridizations, if any.
The reference sample is a mixture of the wild-type samples from different stages.
·	Hybridization design: if applicable, a description of the comparisons made in each hybridization, whether to a standard reference sample, or between experimental samples. An accompanying diagram or table may be useful.
See above.
·	Quality control steps taken: for example, replicates or dye swaps.
No dye swaps were used.  Three replicates in total.
·	URL of any supplemental websites or database accession numbers
Samples used, extract preparation and labeling:
·	The origin of the biological sample (for instance, name of the organism, the provider of the sample) and its characteristics: for example, gender, age, developmental stage, strain, or disease state.  See above.  Tissues/Organs were dissected from at least 12 individuals for each sample.
·	Manipulation of biological samples and protocols used: for example, growth conditions, treatments, separation techniques.  Animals were grown on standard corn meal medium with 0.05% bromophenol blue at 25 degrees Celsius.
·	Protocol for preparing the hybridization extract: for example, the RNA or DNA extraction and purification protocol
Total RNA was extracted using TriZOL.  2 ug of total RNA from each sample was amplified with T7 RNA polymerase using a one-round linear amplification protocol.  RNA extraction and amplification were according to Baugh, L. R., Hill, A. A., Brown, E. L., and Hunter, C. P. (2001). Quantitative analysis of mRNA amplification by in vitro transcription. Nucleic Acids Res 29, E29.
·	Labeling protocol(s).
Priming step with random hexamers.  Labeling step using Superscript II, buffer, DTT, dUTP-Cy3, dUTP-Cy5 and free nucleotides.  Cleanup using Qiagen PCR cleanup kit.  Hybridization to slide in a TE, SSC, SDS solution with poly-A as a blocker.  Hybridization chambers from DIE-TECH, incubated for 10 minutes at 42 degrees followed by approximately 15-hour incubation at 64 degrees.  Three washes with successively more dilute solutions of SSC, including SDS in the first wash, then a centrifuge spin to dry.  Arrays were stored in the dark until scanned.  See K.P. White and K.C. Burtis “Drosophila microarrays: from arrayer construction to hybridization” in Drosophila Protocols, Cold Spring Harbor Press, 2000 for microarray protocols.
·	External controls (spikes).
Hybridization procedures and parameters:
·	The protocol and conditions used during hybridization, blocking and washing.
See above.
Measurement data and specifications:
·	The quantitations based on the images.
Images were gridded and spots were quantified with GenePix software (Axon Instruments)
·	The set of quantitations from several arrays upon which the authors base their conclusions. While access to images of raw data is not required (although its value is unquestionable), authors should make every effort to provide the following:
o	Type of scanning hardware and software used: this information is appropriate for a materials and methods section.
Slides were scanned on an Axon 4000B scanner using Genepix Pro 3.0 software.
o	Type of image analysis software used: specifications should be stated in the materials and methods.
The program GenePix Pro 3.0 from Axon Instruments was used to analyse the data.
o	A description of the measurements produced by the image-analysis software and a description of which measurements were used in the analysis.
(F635 Median - B635), (F532 Median - B532), and (Ratio of Medians) were the raw measurements produced by the software.  The detailed information for these measurements is available at http://www.axon.com/manuals/GenePix_Pro_4.0_User_Guide_Rev_E.pdf
o	The complete output of the image analysis before data selection and transformation (spot quantitation matrices).
See *.xls files attached.
o	Data selection and transformation procedures.
Data for all spots are filtered.  For a spot to pass quality control, 3 conditions need to be met: (1). There is no automatic or manual flag during image gridding using GenePix Pro software.  2. For this spot, at least 1 channel is of at least 75% of the pixels are one SD above background.  3. The net signal noise ratio should be at least 1 for at least one channel.  For the channel 635 nm wavelength, the signal noise ratio is the ratio between (F635_median-B635_median) to the background median.  The background median is the bigger of the median of B635-median of all the spots on the whole array, and B-median of this spot.  For the channel 532 nm wavelength, F532_median and B532_median are used.  Ratios between net signals are log2 transformed and analyzed as detailed in the paper.
o	Final gene expression data table(s) used by the authors to make their conclusions after data selection and transformation (gene expression data matrices).
See the web tables attached for the paper.  Additionally, see the column for Passed and normalized ratio of median in the *.xls files attached.  Data before and after data selection is in one file.
Array Design:
·	General array design, including the platform type (whether the array is a spotted glass array, an in situ synthesized array, etc.); surface and coating specifications (when known – often commercial suppliers do not provide this data); and the availability of the array (the name or make of commercially available arrays).
Spotted array using PCR fragments of approximately 95% of the genes in the D. melanogaster BDGP genome.  Slides were coated with polylysine, printed using a GeneMachines OmniGrid printer with DNA suspended in 3X SSC, and postprocessed using the DCE protocol of Diehl, F., Grahlmann, S., Beier, M. & Hoheisel, J.D. “Manufacturing DNA microarrays of high spot homogeneity and reduced background signal.” Nucleic Acids Research 29, e38 (2001).
·	For each feature (spot) on the array, its location on the array and the ID of its respective reporter (molecule present on each spot) should be given.
The KP numbers identify these and can be tied to the data files.  The ID of the respective reporter is included in the Excel file Web Table PCR Primer and Amplicon Sequence.xls.
·	For each reporter, its type (e.g., cDNA or oligonucleotide) should be given, along with information that characterizes the reporter molecule unambiguously, in the form of appropriate database reference(s) and sequence (if available).
The FBGN and CG n

source_name: 635nm Ch1, the midgut from EcR mutants vs. a reference sample.  EcR mutants (EcRM554fs/EcRV559fs) lacking the function of all EcR isoforms were collected at stage 2a

source_name: 532nm Ch2, the reference sample - wild type (Canton S) midgut samples at various stages


title: SL2_vs_Kc167_S1_1

description:
The expression profiles of Drosophila Kc167 and SL2 cell lines were compared.  Total RNA was extracted from dense cultures of Kc167 or SL2 cells which were maintained at 22C using Trizol Reagent (Invitrogen, Burlington, ON).   Kc167 cells were grown in Schneider's Complete Drosophila Medium (Invitrogen) supplemented with 5% Fetal Bovine Serum (Invitrogen) and 20ug/mL Gentamicin (Sigma, Oakville, ON) whereas SL2 cells were grown in CCM3 media (HyClone, Logan, UT) supplemented with 20ug/mL Gentamicin.  80ug of RNA, quantified by A260 in an Ultrmark plate reader (Biorad, Mississauga, ON), was used in each direct labeling reaction (see http://www.flyarrays.com) to generate fluorescently labeled cDNA.  The respective Cy3 (SL2) and Cy5 (Kc167) targets were suspended in 75 uL DIG EasyHyb solution (Roche, Mississauga, ON)and were competitively hybridized to the array for 16 hours in a CMT-GAPS hybridization chamber (Corning, Acton, MA).  Scanning was performed with a ScanArray 4000 system (Perkin Elmer, Boston, MA) using the following settings:  10uM scan:  Cy3(543 nm) 100-72, Cy5(633 nm) 95-60 (laser power-PMT Gain).  Quantification was performed using QuantArray v3 software (Perkin Elmer) using the adaptive algorithm without background subtraction.  The data in the file is not normalized.  The ratio in the Value column was derived from normalized data (not shown) as discussed in the manuscript.
Keywords = Kc167
Keywords = SL2
Keywords = Drosophila
Lot batch = d7k2-070802


title: SL2_vs_Kc167_S2_flip_1


title: Kc167_Homotypic_R2_1

description:
Total RNA was extracted from a dense culture of Kc167 cells which was maintained at 22C in Schneider's Complete Drosophila Medium (Invitrogen, Burlington, ON) supplemented with 5% Fetal Bovine Serum (Invitrogen) and 20ug/mL Gentamicin (Sigma, Oakville, ON) using Trizol Reagent (Invitrogen).  80ug of RNA, quantified by A260 in an Ultrmark plate reader (Biorad, Mississauga, ON), was used in each direct labeling reaction (see http://www.flyarrays.com) to generate fluorescently labeled cDNA.  The respective Cy3 and Cy5 targets were suspended in 75 uL DIG EasyHyb solution (Roche, Mississauga, ON)and were competitively hybridized to the array for 16 hours in a CMT-GAPS hybridization chamber (Corning, Acton, MA).  Scanning was performed with a ScanArray 4000 system (Perkin Elmer, Boston, MA) using the following settings:  10uM scan:  Cy3(543 nm) 100-72, Cy5(633 nm) 100-62 (laser power-PMT Gain).  Quantification was performed using QuantArray v3 software (Perkin Elmer) using the adaptive algorithm without background subtraction.  The data in the file is not normalized.  The ratio in the Value column was derived from normalized data (not shown) as discussed in the manuscript.
Keywords = Kc167
Keywords = Homotypic
Keywords = Drosophila
Lot batch = d7k2-070802


title: SL2_vs_Kc167_S3_1


title: Ova C3H 1 vs control C3H pool

description: Amplified RNA from lungs of ovalbumen sensitized and challenged C3H 1 mouse vs. pool of amplified RNA from lungs of control C3H mice.

source_name: Ova C3H 1


title: Kc167_Homotypic_R3_1

description:
Total RNA was extracted from a dense culture of Kc167 cells which was maintained at 22C in Schneider's Complete Drosophila Medium (Invitrogen, Burlington, ON) supplemented with 5% Fetal Bovine Serum (Invitrogen) and 20ug/mL Gentamicin (Sigma, Oakville, ON) using Trizol Reagent (Invitrogen).  40ug of RNA, quantified by A260 in an Ultrmark plate reader (Biorad, Mississauga, ON), was used in each direct labeling reaction (see http://www.flyarrays.com) to generate fluorescently labeled cDNA.  The respective Cy3 and Cy5 targets were suspended in 75 uL DIG EasyHyb solution (Roche, Mississauga, ON)and were competitively hybridized to the array for 16 hours in a CMT-GAPS hybridization chamber (Corning, Acton, MA).  Scanning was performed with a ScanArray 4000 system (Perkin Elmer, Boston, MA) using the following settings:  10uM scan:  Cy3(543 nm) 100-72, Cy5(633 nm) 100-62 (laser power-PMT Gain).  Quantification was performed using QuantArray v3 software (Perkin Elmer) using the adaptive algorithm without background subtraction.  The data in the file is not normalized.  The ratio in the Value column was derived from normalized data (not shown) as discussed in the manuscript.
Keywords = Kc167
Keywords = Homotypic
Keywords = Drosophila
Lot batch = d7k2-070802


title: High Fat Mouse 1

status: Public on May 16 2003

city: Cambridge

first: R.

middle: Michael

last: Raab

state: MA

address: 77 Massachusetts Avenue

department: Chemical Engineering

institute: Massachusetts Institute of Technology

description:
A control group consisting of nine C57/BL/6J mice was maintained on a normal diet consisting of Purina Rodent Chow (Harlan Teklad, catalog #5008, 6.5% fat, 49% carbohydrate, 23% protein, 3.5 kcal/ g). The experimental group consisted of nine mice grown on a specialty high-fat diet (Harlan Teklad, catalog #TD88137, 42.16% fat, 42.81% carbohydrate, 15.02% protein, 4.53 kcal/ g). Mice were maintained for ten weeks on their respective diets in a controlled environment with standard light and feeding schedule.  Two days before tissue harvest, the experimental group on the high-fat diet was divided into one group of five and one group of four mice. The first group continued on the high-fat diet for the final two days, while the second group fasted. Mouse weights were recorded two days prior, and the day of tissue harvest. At the end of the fasting period liver tissue samples were harvested from each mouse.

RNA was purified from the liver tissue samples. Each tissue was homogenized in 1 mL of STAT-60 (Tel-Test, Inc., Friendswood, TX) and the homogenate was mixed with 200 uL of chloroform. The aqueous phase was removed, and mixed with 500 uL of isopropanol to precipitate the RNA. The resulting RNA pellet was washed with 500 uL of 70% ethanol and allowed to air dry. The pellet was dissolved in RNase free water (Ambion, TX), quantified by UV spectroscopy, and stored at -80C.

Total RNA control samples (Universal Mouse Reference RNA, catalog # 740100, Stratagene) were labeled using Cy3 dCTP (Perkin-Elmer), and liver total RNA experimental samples were labeled using Cy5 dCTP (Perkin-Elmer) during reverse transcription. For reverse transcription 10 ug of total RNA was mixed with 2 uL of 10X Cy-labeled dCTP, 2 uL of 100 mM DTT (Invitrogen), 4 uL of 5X First Strand Buffer (Invitrogen), 2 uL of a dNTP mixture containing 5 mM dGTP, dATP, dTTP and 2 mM dCTP (Invitrogen), 2 uL of 0.5 mg/ mL oligo-dT18-20 (Invitrogen), and 2 uL of Superscript II reverse transcriptase (Invitrogen). The control RNA samples were additionally mixed with 2 uL of random hexamers. The reaction mixtures were incubated at 42 C for two hours. After the reverse transcription reactions, the RNA templates were degraded by addition of 2 uL of 1 N NaOH and incubation at 65 C for 10 minutes. The alkaline conditions were neutralized by addition of 2 uL of 1 N HCl. Liver cDNA samples were then mixed with a control cDNA sample, and the mixtures were cleaned using a QIAquick Nucleotide Removal kit (Qiagen) as described by the manufacturer's instructions. The cleaned samples were concentrated in a speed vacufuge (Eppendorf) for 20 minutes at 65 C, and were dissolved in 32 uL of prewarmed hybridization buffer (Clontech Laboratories), quantified, and hybridized to prepared oligonucleotide microarrays. Hybridization occurred overnight at 55 C using Corning hybridization chambers. The following day the arrays were washed in Glass Hybridization Wash Solution (Clontech Laboratories), then in 1X SSC, and finally in 0.1X SSC. The arrays were dried by centrifugation and scanned using a GenePix 4000B scanner (Axon Instruments). The resulting data was downloaded and formatted in Excel (Microsoft), and analyzed using Matlab (The MathWorks, Inc.).

Microarrays were prepared using GAPS glass slides (Corning), and a Virtek arrayer (Bio-Rad). Arrays contained 16,847 features, printed from a synthesized oligonucleotide mouse library (Operon). The library was suspended in 3X SSC, centrifuged, and loaded into the arrayer. Arrays were printed at approximately 50% humidity. Once printed, the probes were crosslinked using a UV Stratalinker (Stratagene). The slides were then
blocked for 15 minutes in a 250 mL mixture containing 15.7 g/ L succinic anhydride (Fluka AG), 239 mL 1-methyl-2-pyrrolidinone (EM Science), and 10.7 mL of 1 M boric acid (Fisher), pH 8.0. They were cleaned by rinsing twice in 250 mL of milli-Q water, followed by 250 mL of 95% ethanol (Pharmco Products). After blocking, they were dried using filtered air and stored in the dark at room temperature.
Keywords = mouse, liver, energy homeostasis, gene expression, microarrays

source_name: RNA isolated from the liver of a C57/BL/J6 mouse fed a high fat diet.

source_name: Control RNA obtained from Stratagene.


title: High Fat Mouse 2


title: Kc167_Homotypic_Q1_1

description:
Total RNA was extracted from a dense culture of Kc167 cells which was maintained at 22C in Schneider's Complete Drosophila Medium (Invitrogen, Burlington, ON) supplemented with 5% Fetal Bovine Serum (Invitrogen) and 20ug/mL Gentamicin (Sigma, Oakville, ON) using Trizol Reagent (Invitrogen).  20ug of RNA, quantified by A260 in an Ultrmark plate reader (Biorad, Mississauga, ON), was used in each direct labeling reaction (see http://www.flyarrays.com) to generate fluorescently labeled cDNA.  The respective Cy3 and Cy5 targets were suspended in 75 uL DIG EasyHyb solution (Roche, Mississauga, ON)and were competitively hybridized to the array for 15.75 hours in a CMT-GAPS hybridization chamber (Corning, Acton, MA).  Scanning was performed with a ScanArray 4000 system (Perkin Elmer, Boston, MA) using the following settings:  10uM scan:  Cy3(543 nm) 95-75, Cy5(633 nm) 95-65 (laser power-PMT Gain).  Quantification was performed using QuantArray v3 software (Perkin Elmer) using the adaptive algorithm without background subtraction.  The data in the file is not normalized.  The ratio in the Value column was derived from normalized data (not shown) as discussed in the manuscript.
Keywords = Kc167
Keywords = Homotypic
Keywords = Drosophila
Lot batch = d7k2-100702


title: High Fat Mouse 4

source_name: RNA isolated from the liver of a mouse fed a high fat diet.


title: Fasted Mouse 2

source_name: RNA isolated from the liver of a C57/BL/J6 mouse fed a high fat diet, followed by a 48 hour fast.


title: Kc167_Homotypic_R1_1


title: Kc167_Homotypic_Q2_1

description:
Total RNA was extracted from a dense culture of Kc167 cells which was maintained at 22C in Schneider's Complete Drosophila Medium (Invitrogen, Burlington, ON) supplemented with 5% Fetal Bovine Serum (Invitrogen) and 20ug/mL Gentamicin (Sigma, Oakville, ON) using Trizol Reagent (Invitrogen).  40ug of RNA, quantified by A260 in an Ultrmark plate reader (Biorad, Mississauga, ON), was used in each direct labeling reaction (see http://www.flyarrays.com) to generate fluorescently labeled cDNA.  The respective Cy3 and Cy5 targets were suspended in 75 uL DIG EasyHyb solution (Roche, Mississauga, ON)and were competitively hybridized to the array for 15.75 hours in a CMT-GAPS hybridization chamber (Corning, Acton, MA).  Scanning was performed with a ScanArray 4000 system (Perkin Elmer, Boston, MA) using the following settings:  10uM scan:  Cy3(543 nm) 95-75, Cy5(633 nm) 95-65 (laser power-PMT Gain).  Quantification was performed using QuantArray v3 software (Perkin Elmer) using the adaptive algorithm without background subtraction.  The data in the file is not normalized.  The ratio in the Value column was derived from normalized data (not shown) as discussed in the manuscript.
Keywords = Kc167
Keywords = homotypic
Keywords = Drosophila
Lot batch = d7k2-100702


title: High Fat Mouse 3

source_name: RNA isolated from the liver of a C57/BL/6 mouse fed a high fat diet.


title: Ova C57 5 vs control C57 pool

description: Amplified RNA from lungs of ovalbumen sensitized and challenged C57 5 mouse vs. pool of amplified RNA from lungs of control C57 mice.

source_name: Ova C57 5

source_name: control C57 pool


title: Kc167_Homotypic_Q3_1

description:
Total RNA was extracted from a dense culture of Kc167 cells which was maintained at 22C in Schneider's Complete Drosophila Medium (Invitrogen, Burlington, ON) supplemented with 5% Fetal Bovine Serum (Invitrogen) and 20ug/mL Gentamicin (Sigma, Oakville, ON) using Trizol Reagent (Invitrogen).  80ug of RNA, quantified by A260 in an Ultrmark plate reader (Biorad, Mississauga, ON), was used in each direct labeling reaction (see http://www.flyarrays.com) to generate fluorescently labeled cDNA.  The respective Cy3 and Cy5 targets were suspended in 75 uL DIG EasyHyb solution (Roche, Mississauga, ON)and were competitively hybridized to the array for 15.75 hours in a CMT-GAPS hybridization chamber (Corning, Acton, MA).  Scanning was performed with a ScanArray 4000 system (Perkin Elmer, Boston, MA) using the following settings:  10uM scan:  Cy3(543 nm) 95-75, Cy5(633 nm) 95-65 (laser power-PMT Gain).  Quantification was performed using QuantArray v3 software (Perkin Elmer) using the adaptive algorithm without background subtraction.  The data in the file is not normalized.  The ratio in the Value column was derived from normalized data (not shown) as discussed in the manuscript.
Keywords = Kc167
Keywords = Homotypic
Keywords = Drosophila
Lot batch = d7k2-100702


title: Ova C3H 4 vs control C3H pool

description: Amplified RNA from lungs of ovalbumen sensitized and challenged C3H 4 mouse vs. pool of amplified RNA from lungs of control C3H mice.

source_name: Ova C3H 4


title: SL2_vs_Kc167_S2_1


title: Control Mouse 4

source_name: RNA isolated from the liver of a C57/BL/J6 mouse fed a normal diet.


title: Control Mouse 3


title: Ova C3H 2 vs control C3H pool

description: Amplified RNA from lungs of ovalbumen sensitized and challenged C3H 2 mouse vs. pool of amplified RNA from lungs of control C3H mice.

source_name: Ova C3H 2


title: Control Mouse 1

status: Public on May 15 2003

source_name: C57/BL/J6 Mice grown on a normal diet.


title: 4mM H2O2:ctrl (2h)_2

status: Public on Apr 18 2003

city: Rockville

first: Helena

middle: I

last: Boshoff

state: MD

address: 12441 Parklawn Drive

department: LIG

institute: NIAID/ NIH

description:
H37Rv treated for 2h with 4mM H2O2 in 7H9/ADC/Tween at OD650 = 0.3 versus control untreated cells
Sample_array_print_batch: Mtaa-04
Sample_image_analysis_software: Axon GenePix Pro
Sample_image_analysis_software_version: 3.0.6.66
Sample_microarray_scanner: GenePix 4000B [82425]
Sample_NIH_mAdb_system_expt_id: 24082
Sample_organism_strain: H37Rv
Sample_treatment_agent: hydrogen peroxide
Sample_treatment_dose: 4
Sample_treatment_dose_unit: mM
Sample_treatment_timepoint: 2
Sample_treatment_timepoint_unit: h
Sample_treatment_vehicle: None
Keywords = DNA damage

organism: Mycobacterium tuberculosis

source_name: control


title: 60mJ/cm2 UV:ctrl (6h)

description:
H37Rv irradiated with UV at 60 mJ/cm2 in open Petri dish (2E+10 cells in 5mL 7H9/ADC/Tween) with 6h recovery in 50mL 7H9/ADC/Tween at 37C versus control unirradiated cells
Sample_array_print_batch: Mtaa-05
Sample_image_analysis_software: Axon GenePix Pro
Sample_image_analysis_software_version: 3.0.6.66
Sample_microarray_scanner: GenePix 4000B [82425]
Sample_NIH_mAdb_system_expt_id: 24094
Sample_organism_strain: H37Rv
Sample_treatment_agent: UV radiation
Sample_treatment_dose: 60
Sample_treatment_dose_unit: mJ/cm2
Sample_treatment_timepoint: 6
Sample_treatment_timepoint_unit: h
Sample_treatment_vehicle: None
Keywords = DNA damage

source_name: UV


title: Control Mouse 2


title: 4mM H2O2:ctrl (4h)_2

description:
H37Rv treated for 4h with 4mM H2O2 at OD650 = 0.3 versus control untreated cells
Sample_array_print_batch: Mtaa-05
Sample_image_analysis_software: Axon GenePix Pro
Sample_image_analysis_software_version: 3.0.6.66
Sample_microarray_scanner: GenePix 4000B [82425]
Sample_NIH_mAdb_system_expt_id: 24086
Sample_organism_strain: H37Rv
Sample_treatment_agent: hydrogen peroxide
Sample_treatment_dose: 4
Sample_treatment_dose_unit: mM
Sample_treatment_timepoint: 4
Sample_treatment_timepoint_unit: h
Sample_treatment_vehicle: None
Keywords = DNA damage


title: 60mJ/cm2 UV:ctrl (2h)

description:
H37Rv irradiated with UV at 60 mJ/cm2 in open Petri dish (2E+10 cells in 5mL 7H9/ADC/Tween) with 2h recovery in 50mL 7H9/ADC/Tween at 37C versus control unirradiated cells
Sample_array_print_batch: Mtaa-05
Sample_image_analysis_software: Axon GenePix Pro
Sample_image_analysis_software_version: 3.0.6.66
Sample_microarray_scanner: GenePix 4000B [82425]
Sample_NIH_mAdb_system_expt_id: 24090
Sample_organism_strain: H37Rv
Sample_treatment_agent: UV radiation
Sample_treatment_dose: 60
Sample_treatment_dose_unit: mJ/cm2
Sample_treatment_timepoint: 2
Sample_treatment_timepoint_unit: h
Sample_treatment_vehicle: None
Keywords = DNA damage


title: 25mJ/cm2 UV:ctrl (4h)

description:
H37Rv irradiated with UV at 25 mJ/cm2 in open Petri dish (2E+10 cells in 5mL 7H9/ADC/Tween) with 4h recovery in 50mL 7H9/ADC/Tween at 37C versus control unirradiated cells
Sample_array_print_batch: Mtaa-05
Sample_image_analysis_software: Axon GenePix Pro
Sample_image_analysis_software_version: 3.0.6.66
Sample_microarray_scanner: GenePix 4000B [82425]
Sample_NIH_mAdb_system_expt_id: 24091
Sample_organism_strain: H37Rv
Sample_treatment_agent: UV radiation
Sample_treatment_dose: 25
Sample_treatment_dose_unit: mJ/cm2
Sample_treatment_timepoint: 4
Sample_treatment_timepoint_unit: h
Sample_treatment_vehicle: None
Keywords = DNA damage


title: 25mJ/cm2 UV:ctrl (2h)

description:
H37Rv irradiated with UV at 25 mJ/cm2 in open Petri dish (2E+10 cells in 5mL 7H9/ADC/Tween) with 2h recovery in 50mL 7H9/ADC/Tween at 37C versus control unirradiated cells
Sample_array_print_batch: Mtaa-05
Sample_image_analysis_software: Axon GenePix Pro
Sample_image_analysis_software_version: 3.0.6.66
Sample_microarray_scanner: GenePix 4000B [82425]
Sample_NIH_mAdb_system_expt_id: 24089
Sample_organism_strain: H37Rv
Sample_treatment_agent: UV radiation
Sample_treatment_dose: 25
Sample_treatment_dose_unit: mJ/cm2
Sample_treatment_timepoint: 2
Sample_treatment_timepoint_unit: h
Sample_treatment_vehicle: None
Keywords = DNA damage


title: Fasted Mouse 1


title: 40mJ/cm2 UV:ctrl (12h)

description:
H37Rv irradiated with UV at 40 mJ/cm2 in open Petri dish (2E+10 cells in 5mL 7H9/ADC/Tween) with 12h recovery in 50mL 7H9/ADC/Tween at 37C versus control unirradiated cells
Sample_array_print_batch: Mtaa-04
Sample_image_analysis_software: Axon GenePix Pro
Sample_image_analysis_software_version: 3.0.6.73
Sample_microarray_scanner: GenePix 4000A [55478]
Sample_NIH_mAdb_system_expt_id: 25503
Sample_organism_strain: H37Rv
Sample_treatment_agent: UV radiation
Sample_treatment_dose: 40
Sample_treatment_dose_unit: mJ/cm2
Sample_treatment_timepoint: 12
Sample_treatment_timepoint_unit: h
Sample_treatment_vehicle: None
Keywords = DNA damage


title: 0.2ug/mL MMC:ctrl (6h)

description:
H37Rv treated for 6h with 0.2ug/mL MMC in 7H9/ADC/Tween at OD650 = 0.3 versus control untreated cells
Sample_array_print_batch: Mtaa-05
Sample_image_analysis_software: Axon GenePix Pro
Sample_image_analysis_software_version: 3.0.6.66
Sample_microarray_scanner: GenePix 4000B [82425]
Sample_NIH_mAdb_system_expt_id: 24079
Sample_organism_strain: H37Rv
Sample_treatment_agent: MMC
Sample_treatment_dose: 0.2
Sample_treatment_dose_unit: microgram/ml
Sample_treatment_timepoint: 6
Sample_treatment_timepoint_unit: h
Sample_treatment_vehicle: DMF (1/10000 dilution )
Keywords = DNA damage

source_name: MMC


title: 0.2ug/mL MMC:ctrl (4h)

description:
H37Rv treated for 4h with 0.2ug/mL MMC in 7H9/ADC/Tween at OD650 = 0.3 versus control untreated cells
Sample_array_print_batch: Mtaa-05
Sample_image_analysis_software: Axon GenePix Pro
Sample_image_analysis_software_version: 3.0.6.66
Sample_microarray_scanner: GenePix 4000B [82425]
Sample_NIH_mAdb_system_expt_id: 24077
Sample_organism_strain: H37Rv
Sample_treatment_agent: MMC
Sample_treatment_dose: 0.2
Sample_treatment_dose_unit: microgram/ml
Sample_treatment_timepoint: 4
Sample_treatment_timepoint_unit: h
Sample_treatment_vehicle: DMF (1/10000 dilution )
Keywords = DNA damage


title: 4mM H2O2:ctrl (12h)

description:
H37Rv treated for 12h with 4mM H2O2 in 7H9/ADC/Tween at OD650 = 0.3 versus control untreated cells
Sample_array_print_batch: Mtaa-04
Sample_image_analysis_software: Axon GenePix Pro
Sample_image_analysis_software_version: 3.0.6.73
Sample_microarray_scanner: GenePix 4000A [55478]
Sample_NIH_mAdb_system_expt_id: 25499
Sample_organism_strain: H37Rv
Sample_treatment_agent: hydrogen peroxide
Sample_treatment_dose: 4
Sample_treatment_dose_unit: mM
Sample_treatment_timepoint: 12
Sample_treatment_timepoint_unit: h
Sample_treatment_vehicle: None
Keywords = DNA damage


title: Fasted Mouse 3

description:
A control group consisting of nine C57/BL/6J mice was maintained on a normal diet consisting of Purina Rodent Chow (Harlan Teklad, catalog #5008, 6.5% fat, 49% carbohydrate, 23% protein, 3.5 kcal/ g). The experimental group consisted of nine mice grown on a specialty high-fat diet (Harlan Teklad, catalog #TD88137, 42.16% fat, 42.81% carbohydrate, 15.02% protein, 4.53 kcal/ g). Mice were maintained for ten weeks on their respective diets in a controlled environment with standard light and feeding schedule.  Two days before tissue harvest, the experimental group on the high-fat diet was divided into one group of five and one group of four mice. The first group continued on the high-fat diet for the final two days, while the second group fasted. Mouse weights were recorded two days prior, and the day of tissue harvest. At the end of the fasting period liver tissue samples were harvested from each mouse.

RNA was purified from the liver tissue samples. Each tissue was homogenized in 1 mL of STAT-60 (Tel-Test, Inc., Friendswood, TX) and the homogenate was mixed with 200 uL of chloroform. The aqueous phase was removed, and mixed with 500 uL of isopropanol to precipitate the RNA. The resulting RNA pellet was washed with 500 uL of 70% ethanol and allowed to air dry. The pellet was dissolved in RNase free water (Ambion, TX), quantified by UV spectroscopy, and stored at -80C.

Total RNA control samples (Universal Mouse Reference RNA, catalog # 740100, Stratagene) were labeled using Cy3 dCTP (Perkin-Elmer), and liver total RNA experimental samples were labeled using Cy5 dCTP (Perkin-Elmer) during reverse transcription. For reverse transcription 10 ug of total RNA was mixed with 2 uL of 10X Cy-labeled dCTP, 2 uL of 100 mM DTT (Invitrogen), 4 uL of 5X First Strand Buffer (Invitrogen), 2 uL of a dNTP mixture containing 5 mM dGTP, dATP, dTTP and 2 mM dCTP (Invitrogen), 2 uL of 0.5 mg/ mL oligo-dT18-20 (Invitrogen), and 2 uL of Superscript II reverse transcriptase (Invitrogen). The control RNA samples were additionally mixed with 2 uL of random hexamers. The reaction mixtures were incubated at 42 C for two hours. After the reverse transcription reactions, the RNA templates were degraded by addition of 2 uL of 1 N NaOH and incubation at 65 C for 10 minutes. The alkaline conditions were neutralized by addition of 2 uL of 1 N HCl. Liver cDNA samples were then mixed with a control cDNA sample, and the mixtures were cleaned using a QIAquick Nucleotide Removal kit (Qiagen) as described by the manufacturer's instructions. The cleaned samples were concentrated in a speed vacufuge (Eppendorf) for 20 minutes at 65 C, and were dissolved in 32 uL of prewarmed hybridization buffer (Clontech Laboratories), quantified, and hybridized to prepared oligonucleotide microarrays. Hybridization occurred overnight at 55 C using Corning hybridization chambers. The following day the arrays were washed in Glass Hybridization Wash Solution (Clontech Laboratories), then in 1X SSC, and finally in 0.1X SSC. The arrays were dried by centrifugation and scanned using a GenePix 4000B scanner (Axon Instruments). The resulting data was downloaded and formatted in Excel (Microsoft), and analyzed using Matlab (The MathWorks, Inc.).

Microarrays were prepared using GAPS glass slides (Corning), and a Virtek arrayer (Bio-Rad). Arrays contained 16,847 features, printed from a synthesized oligonucleotide mouse library (Operon). The library was suspended in 3X SSC, centrifuged, and loaded into the arrayer. Arrays were printed at approximately 50% humidity. Once printed, the probes were crosslinked using a UV Stratalinker (Stratagene). The slides were then
blocked for 15 minutes in a 250 mL mixture containing 15.7 g/ L succinic anhydride (Fluka AG), 239 mL 1-methyl-2-pyrrolidinone (EM Science), and 10.7 mL of 1 M boric acid (Fisher), pH 8.0. They were cleaned by rinsing twice in 250 mL of milli-Q water, followed by 250 mL of 95% ethanol (Pharmco Products). After blocking, they were dried using filtered air and stored in the dark at room temperature.
Keywords = mouse, liver, energy homeostasis, gene expression, microarrays


title: 0.2ug/mL MMC:ctrl (8h)

description:
H37Rv treated for 8h with 0.2ug/mL MMC in 7H9/ADC/Tween at OD650 = 0.3 versus control untreated cells
Sample_array_print_batch: Mtaa-04
Sample_image_analysis_software: Axon GenePix Pro
Sample_image_analysis_software_version: 3.0.6.73
Sample_microarray_scanner: GenePix 4000A [55478]
Sample_NIH_mAdb_system_expt_id: 25444
Sample_organism_strain: H37Rv
Sample_treatment_agent: MMC
Sample_treatment_dose: 0.2
Sample_treatment_dose_unit: microgram/ml
Sample_treatment_timepoint: 8
Sample_treatment_timepoint_unit: h
Sample_treatment_vehicle: DMF (1/10000 dilution )
Keywords = DNA damage


title: 0.2ug/mL MMC:ctrl (12h)

description:
H37Rv treated for 12 h with 0.2ug/mL MMC in 7H9/ADC/Tween at OD650 = 0.3 versus control untreated cells
Sample_array_print_batch: Mtaa-04
Sample_image_analysis_software: Axon GenePix Pro
Sample_image_analysis_software_version: 3.0.6.73
Sample_microarray_scanner: GenePix 4000A [55478]
Sample_NIH_mAdb_system_expt_id: 25497
Sample_organism_strain: H37Rv
Sample_treatment_agent: MMC
Sample_treatment_dose: 0.2
Sample_treatment_dose_unit: microgram/ml
Sample_treatment_timepoint: 12
Sample_treatment_timepoint_unit: h
Sample_treatment_vehicle: DMF (1/10000 dilution )
Keywords = DNA damage


title: 20mJ/cm2 UV:ctrl (8h)

description:
H37Rv irradiated with UV at 20 mJ/cm2 in open Petri dish (2E+10 cells in 5mL 7H9/ADC/Tween) with 8h recovery in 50mL 7H9/ADC/Tween at 37C versus control unirradiated cells
Sample_array_print_batch: Mtaa-04
Sample_image_analysis_software: Axon GenePix Pro
Sample_image_analysis_software_version: 3.0.6.73
Sample_microarray_scanner: GenePix 4000A [55478]
Sample_NIH_mAdb_system_expt_id: 25463
Sample_organism_strain: H37Rv
Sample_treatment_agent: UV radiation
Sample_treatment_dose: 20
Sample_treatment_dose_unit: mJ/cm2
Sample_treatment_timepoint: 8
Sample_treatment_timepoint_unit: h
Sample_treatment_vehicle: None
Keywords = DNA damage


title: 4mM H2O2:ctrl (8h)

description:
H37Rv treated for 8h with 4mM H2O2 in 7H9/ADC/Tween at OD650 = 0.3 versus control untreated cells
Sample_array_print_batch: Mtaa-04
Sample_image_analysis_software: Axon GenePix Pro
Sample_image_analysis_software_version: 3.0.6.73
Sample_microarray_scanner: GenePix 4000A [55478]
Sample_NIH_mAdb_system_expt_id: 25458
Sample_organism_strain: H37Rv
Sample_treatment_agent: hydrogen peroxide
Sample_treatment_dose: 4
Sample_treatment_dose_unit: mM
Sample_treatment_timepoint: 8
Sample_treatment_timepoint_unit: h
Sample_treatment_vehicle: None
Keywords = DNA damage


title: 20mJ/cm2 UV:ctrl (12h)

description:
H37Rv irradiated with UV at 20 mJ/cm2 in open Petri dish (2E+10 cells in 5mL 7H9/ADC/Tween) with 12h recovery in 50mL 7H9/ADC/Tween at 37C versus control unirradiated cells
Sample_array_print_batch: Mtaa-04
Sample_image_analysis_software: Axon GenePix Pro
Sample_image_analysis_software_version: 3.0.6.73
Sample_microarray_scanner: GenePix 4000A [55478]
Sample_NIH_mAdb_system_expt_id: 25500
Sample_organism_strain: H37Rv
Sample_treatment_agent: UV radiation
Sample_treatment_dose: 20
Sample_treatment_dose_unit: mJ/cm2
Sample_treatment_timepoint: 6
Sample_treatment_timepoint_unit: h
Sample_treatment_vehicle: None
Keywords = DNA damage


title: 20mJ/cm2 UV: ctrl (6h)

description:
H37Rv irradiated with UV at 20 mJ/cm2 in open Petri dish (2E+10 cells in 5mL 7H9/ADC/Tween) with 6h recovery in 50mL 7H9/ADC/Tween at 37C versus control unirradiated cells
Sample_array_print_batch: Mtaa-04
Sample_image_analysis_software: Axon GenePix Pro
Sample_image_analysis_software_version: 3.0.6.73
Sample_microarray_scanner: GenePix 4000A [55478]
Sample_NIH_mAdb_system_expt_id: 25424
Sample_organism_strain: H37Rv
Sample_treatment_agent: UV radiation
Sample_treatment_dose: 20
Sample_treatment_dose_unit: mJ/cm2
Sample_treatment_timepoint: 6
Sample_treatment_timepoint_unit: h
Sample_treatment_vehicle: None
Keywords = DNA damage


title: 40mJ/cm2 UV:ctrl (6h)

description:
H37Rv irradiated with UV at 40 mJ/cm2 in open Petri dish (2E+10 cells in 5mL 7H9/ADC/Tween) with 6h recovery in 50mL 7H9/ADC/Tween at 37C versus control unirradiated cells
Sample_array_print_batch: Mtaa-04
Sample_image_analysis_software: Axon GenePix Pro
Sample_image_analysis_software_version: 3.0.6.73
Sample_microarray_scanner: GenePix 4000A [55478]
Sample_NIH_mAdb_system_expt_id: 25440
Sample_organism_strain: H37Rv
Sample_treatment_agent: UV radiation
Sample_treatment_dose: 40
Sample_treatment_dose_unit: mJ/cm2
Sample_treatment_timepoint: 6
Sample_treatment_timepoint_unit: h
Sample_treatment_vehicle: None
Keywords = DNA damage


title: 4mM H2O2:ctrl (2h)

description:
H37Rv treated for 2h with 4mM H2O2 in 7H9/ADC/Tween  at OD650 = 0.3 versus control untreated cells
Sample_array_print_batch: Mtaa-03
Sample_image_analysis_software: Axon GenePix Pro
Sample_image_analysis_software_version: 3.0.6.66
Sample_microarray_scanner: GenePix 4000B [82425]
Sample_NIH_mAdb_system_expt_id: 24081
Sample_organism_strain: H37Rv
Sample_treatment_agent: hydrogen peroxide
Sample_treatment_dose: 4
Sample_treatment_dose_unit: mM
Sample_treatment_timepoint: 2
Sample_treatment_timepoint_unit: h
Sample_treatment_vehicle: None
Keywords = DNA damage


title: 4mM H2O2:ctrl (4h)

description:
H37Rv treated for 4h with 4mM H2O2 in 7H9/ADC/Tween at OD650 = 0.3 versus control untreated cells
Sample_array_print_batch: Mtaa-03
Sample_image_analysis_software: Axon GenePix Pro
Sample_image_analysis_software_version: 3.0.6.66
Sample_microarray_scanner: GenePix 4000B [82425]
Sample_NIH_mAdb_system_expt_id: 24085
Sample_organism_strain: H37Rv
Sample_treatment_agent: hydrogen peroxide
Sample_treatment_dose: 4
Sample_treatment_dose_unit: mM
Sample_treatment_timepoint: 4
Sample_treatment_timepoint_unit: h
Sample_treatment_vehicle: None
Keywords = DNA damage