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
X-coordinate of the center of the feature-indicator associated with the feature, where (0,0) is the top left of the image
Y
Y-coordinate of the center of the feature-indicator associated with the feature, where (0,0) is the top left of the image
Dia.
Diameter in µm of the feature-indicator
F635 Median
Median pixel intensity of feature for Cy5 channel
F635 Mean
Mean pixel intensity of feature for Cy5 channel
F635 SD
Cy5 pixel intensity standard deviation
B635 Median
Median Cy5 feature background intensity
B635 Mean
Mean Cy5 feature background intensity
B635 SD
Cy5 background pixel intensity standard deviation
% > B635+1SD
Percentage of feature pixels with intensities more than one standard deviation above the background pixel intensity, at wavelength #1 (635 nm, Cy5)
% > B635+2SD
Percentage of feature pixels with intensities more than two standard deviations above the background pixel intensity, at wavelength #1 (635 nm, Cy5)
F635 % Sat.
Percentage of pixel saturation within feature
F532 Median
Median feature pixel intensity at wavelength #2 (532 nm, Cy3)
F532 Mean
Mean feature pixel intensity at wavelength #2 (532 nm, Cy3)
F532 SD
Standard deviation of the feature pixel intensity at wavelength #2 (532 nm, Cy3)
B532 Median
Median feature background intensity at wavelength #2 (532 nm, Cy3)
B532 Mean
Mean feature background intensity at wavelength #2 (532 nm, Cy3)
B532 SD
Standard deviation of the feature background intensity at wavelength #2 (532 nm, Cy3)
% > B532+1SD
Percentage of feature pixels with intensities more than one standard deviation above the background pixel intensity, at wavelength #2 (532 nm, Cy3)
% > B532+2SD
Percentage of feature pixels with intensities more than two standard deviations above the background pixel intensity, at wavelength #2 (532 nm, Cy3)
F532 % Sat.
Percentage of feature pixels at wavelength #2 (Cy3) that are saturated
Ratio of Medians
Ratio of the background subtracted median pixel intensity at the second wavelength (Cy3), to the background subtracted median pixel intensity at the first wavelength(Cy5)
Ratio of Means
Ratio of the arithmetic mean of the background subtracted raw pixel intensities at the second wavelength (Cy3), to the arithmetic mean of the background subtracted raw pixel intensities at the first wavelength (Cy5)
Median of Ratios
Median of the pixel-by-pixel ratios of pixel intensities that have had the median background intensity subtracted of wavelength 2 (Cy3) to wavelength one (Cy5)
Mean of Ratios
Arithmetic mean of the pixel-by-pixel ratios of the raw pixel intensities of wavelength 2 (Cy3) to wavelength 1 (Cy5)
Ratios SD
Standard deviation of the log of pixel intensity ratios
Rgn Ratio
Regression ratio is determined by computing a linear regression between the population of pixels represented by wavelength 1 and wavelength 2
Rgn R²
Coefficient of determination provides a measure of the level of accuracy of the fit of the linear regression curve
F Pixels
Number of feature pixels
B Pixels
Number of background pixels
Sum of Medians
Sum of the median of the pixel intensities at each wavelength, with the median background pixel intensity at each wavelength subtracted
Sum of Means
Sum of the arithmetic mean of the pixel intensities at each wavelength, with the median background pixel intensity at each wavelength subtracted
Log Ratio
Log Ratio
F635 Median - B635
Cy5 median feature intensity subtracted by Cy5 median background intensity
F532 Median - B532
Cy3 median feature intensity subtracted by Cy3 median background intensity
F635 Mean - B635
Cy5 mean feature intensity subtracted by Cy5 mean background intensity
F532 Mean - B532
Cy3 mean feature intensity subtracted by Cy3 mean background intensity
Flags
A value of -100 means that this spot was bad. A value of -75 means that the ID for this spot is empty. A value of -50 means that this spot cannot be aligned during analysis. A value of 0 or larger than 0 means that this spot passed the flagging screen but may still fail other quality tests.
NormFactorMedian
The normalization factor calculated using Ratio of Medians
VALUE
same as UNF_VALUE but with flagged values removed
QUALITY
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) 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 532 nm wavelength, F532_median and B532_median are used.
PRE_VALUE
The product of Ratio of Medians and NormFactorMedian
