Flies were grown at 25°C on PB medium (KD Medical, Columbia, MD) for 3 to 5 days post eclosion and mated. Embryos were then collected and quick frozen on dry ice and total RNA was extracted (Andrews et al, Genome Research 10:2030-2043) using Trizol reagent (GibcoBRL, Gaithersburg, MD).
Extracted molecule
total RNA
Extraction protocol
Embryos collected and quick frozen on dry ice and total RNA was extracted (Andrews et al, Genome Research 10:2030-2043) using Trizol reagent (GibcoBRL, Gaithersburg, MD).
Label
Cy3
Label protocol
Probes were labeled with Cy3 (Probe 1) or Cy5 (Probe 2). To synthesize probes, mRNA was isolated by a single round of poly(A) selection using Oligotex resin (Qiagen, Valencia, CA). The purified mRNA was quantified using RiboGreen dye (Molecular Probes) in a fluorescent assay. RiboGreen dye was diluted 1:200 (v/v final) and mixed with known RNA concentrations (determined by absorbance at 260 nm) ranging from 1 to 5000 ng/ml. A Millennium RNA size ladder (Ambion, Austin, TX) was used to generate standard curves and unknown samples were diluted as necessary. Fluorescence was measured in 96-well plates with a FLUOstar fluorometer (BMG Lab Technologies, Germany) fitted with 485 nm (excitation) and 520 nm (emission) filters. Between 25 and 100 ng mRNA were separated on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA), to examine the mRNA size distribution. 200 ng of purified mRNA were converted to either a Cy3- or Cy5-labeled cDNA probe using a custom labeling kit (Incyte Genomics). Each reaction contained 50 mM Tris-HCl pH 8.3, 75 mM KCl, 15 mM MgCl2, 4 mM DTT, 2 mM dNTPs (0.5 mM each), 2 µg Cy3 or Cy5 random 9mer (Trilink, San Diego, CA), 20 U RNase inhibitor (Ambion), 200 U MMLV RNase H-free reverse transcriptase (Promega, Madison, WI) and mRNA. Correspondingly labeled Cy3 and Cy5 cDNA products were combined and purified on a size exclusion column, concentrated by ethanol precipitation and resuspended in hybridization buffer.
Flies were grown at 25°C on PB medium (KD Medical, Columbia, MD) for 3 to 5 days post eclosion and mated. Embryos were then collected and quick frozen on dry ice and total RNA was extracted (Andrews et al, Genome Research 10:2030-2043) using Trizol reagent (GibcoBRL, Gaithersburg, MD).
Extracted molecule
total RNA
Extraction protocol
Embryos collected and quick frozen on dry ice and total RNA was extracted (Andrews et al, Genome Research 10:2030-2043) using Trizol reagent (GibcoBRL, Gaithersburg, MD).
Label
Cy5
Label protocol
Probes were labeled with Cy3 (Probe 1) or Cy5 (Probe 2). To synthesize probes, mRNA was isolated by a single round of poly(A) selection using Oligotex resin (Qiagen, Valencia, CA). The purified mRNA was quantified using RiboGreen dye (Molecular Probes) in a fluorescent assay. RiboGreen dye was diluted 1:200 (v/v final) and mixed with known RNA concentrations (determined by absorbance at 260 nm) ranging from 1 to 5000 ng/ml. A Millennium RNA size ladder (Ambion, Austin, TX) was used to generate standard curves and unknown samples were diluted as necessary. Fluorescence was measured in 96-well plates with a FLUOstar fluorometer (BMG Lab Technologies, Germany) fitted with 485 nm (excitation) and 520 nm (emission) filters. Between 25 and 100 ng mRNA were separated on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA), to examine the mRNA size distribution. 200 ng of purified mRNA were converted to either a Cy3- or Cy5-labeled cDNA probe using a custom labeling kit (Incyte Genomics). Each reaction contained 50 mM Tris-HCl pH 8.3, 75 mM KCl, 15 mM MgCl2, 4 mM DTT, 2 mM dNTPs (0.5 mM each), 2 µg Cy3 or Cy5 random 9mer (Trilink, San Diego, CA), 20 U RNase inhibitor (Ambion), 200 U MMLV RNase H-free reverse transcriptase (Promega, Madison, WI) and mRNA. Correspondingly labeled Cy3 and Cy5 cDNA products were combined and purified on a size exclusion column, concentrated by ethanol precipitation and resuspended in hybridization buffer.
Hybridization protocol
Probes were added between two subarray slides and developed as described (Yue et al.2001, NAR. 29:e41).
Scan protocol
Microarrays were scanned on a GenePix 4000A scanner (Axon Instruments, Foster City, CA). Signal intensities were initially captured using GenePix Pro 4.1 (Axon Instruments, Foster City CA).
Description
Menin overexpression mutants were generated using transgenic lines bearing the full-length Mnn1 cDNA (Guru et al., 2001) under the control of the yeast GAL4 UAS promoter (Brand and Perrimon, 1993). The constructs were introduced into flies by P-element mediated transgenesis (Rubin and Spradling, 1982). Mutants are described in Cerrato et al., Dev Biol. 2006 Oct 1;298(1):59-70. Control samples are flies that have the same driver, but do not have the without the Mnn1 overexpressing P-element construct.
Data processing
Normalization by within-slide print tip loess and between-slide quantile, along with subsequent analyses, were performed using the bioconductor package LIMMA 2.12.0 (Smyth et.al, 2004).
Log2 transformed intensity signal from Cy3 channel. Values are normalized within each array by print tip Loess and across the series using quantile normalization in Bioconductor.
Log2Cy5
Log2 transformed intensity signal from Cy5 channel. Values are normalized within each array by print tip Loess and across the series using quantile normalization in Bioconductor.
VALUE
Log2 transformed ratio of corrected Cy3/Cy5 signal calculated by taking the corrected Log2Cy3 signal value and subtracting the Log2Cy5 signal value for each element
Cy3_SIGNAL
Raw median signal intensity data from from Cy3 channel acquired by Genepix
Cy5_SIGNAL
Raw median signal intensity data from from Cy5 channel acquired by Genepix
INV_VALUE
Log2 transformed ratio of corrected Cy5/Cy3 signal calculated by taking the corrected Log2Cy5 signal value and subtracting the Log2Cy3 signal value for each element
