title: Transdetermination study; regenerating discs vs reference_5-189

description: These experiments are described in Klebes et al. (Development, 2005, in press). Comparisons – category #9, slide 5-189, reference sample – probe 1 vs. regenerating disc – probe 2; (ratios are shown as probe1/probe2). In this experiment, regenerating disc cells were compared to the common leg disc reference sample. These comparisons were intended to establish the unique expression profiles of regenerating leg imaginal cells in response to fragmentation. Regenerating cells were obtained from disc fragments that had been cultured in the abdomen of female flies to promote proliferation. To minimize possible contamination with transdetermined cells, we did not use 1st leg discs, but instead examined the ‘3/4 fragment’ of 2nd leg discs (Sustar and Schubiger, 2005). This fragment regenerates missing parts, and in contrast to 1st leg discs, does not transdetermine (G.S., unpublished). The disc fragments were collected after 3-5 days of culture. The common reference sample was generated by pooling amplified RNA of male and female 1st, 2nd and 3rd leg imaginal discs in equal proportions. Discs and fragments were dissected in PBS and carefully washed several times to reduce the concentration of contaminating fat body cells. RNA was extracted from imaginal disc fragments using the Mini RNA isolation kit (Zymo research). Linear RNA amplification was performed essentially as described (Klebes et al., 2002). In brief, total RNA was used for a first round of reverse transcription and in vitro transcription using T7 RNA polymerase. Subsequently, the amplified RNA product was subjected to a 2nd round of reverse transcription and in vitro transcription yielding 10,000-fold amplification or more. Probes were indirectly labeled with amino-allyl modified dUTP in a reverse transcription reaction and coupled to reactive Cy3 or Cy5 dyes (Amersham). Correspondingly labeled Cy3 and Cy5 cDNA products were combined and purified on PCR purification columns (Qiagen), and diluted in hybridization buffer. Probes were added to the slides and incubated in a metal hybidization chamber (DieTech) in a waterbath at 65°C over night. Microarrays were washed and scanned on a GenePix 4000A scanner (Axon Instruments, Foster City, CA). Signal intensities were initially stored and analyzed using NOMAD (www.microarrays.org). Data was extracted as the global median normalized ratios (net median ratios) of the intensitites and log2 transformed. Signals with intensity values of the sum of median (foreground minus background) smaller than 300 were eliminated. Each comparison was performed in replicates with independently isolated and processed probes. Within a set of replicate experiments, dyes were reversed to avoid bias in about half of the experiments. To represent all experiments with the same dye orientation the values of the log2 transformed expression ratios were reversed if necessary.

data_processing: log2 transformed global median normalized ratios (probe1/probe2)

tag: Cultivated 3/4 fragment of a mesothoracic (2nd) leg imaginal disc, 2 rounds of linear RNA amplificatio

source_name: leg disc reference sample


title: Transdetermination study; D(wt) cells vs V(wt) cells_7-020

status: Public on Jul 08 2005

type: RNA

city: Berlin

first: Ansgar

last: Klebes

address: Chausseestr. 117

department: Biology

country: Germany

institute: Humboldt University Berlin

description: These experiments are described in Klebes et al. (Development, 2005, in press).  Comparisons – category #8,  slide 7-020, D(wt),  V(wt) ventral cells from the same discs– probe 1 vs. dorsal prothoracic leg disc cells (wildtype) – probe 2; (ratios are shown as probe 1/probe 2).  Dorsal cells (D(wt) of prothoracic leg imaginal discs were compared to ventral cells (V(wt)). Both cell populations were isolated from the same discs. For this experiment third instar larvae of Drosophila melanogaster strain OregonR (OR) were staged by feeding 1% bromphenol blue which allows to observe the gut content. Single male wandering late third instar larvae (half empty gut) were used to isolate leg discs. These comparisons were intended to establish the unique expression profiles of dorsal and ventral leg imaginal cells to distinguish them from genes identified in the transdeterminating cell population of Wingless over-expressing leg discs. Dorsal cells of wildtype prothoracic leg discs (D(wt)) were approximately separated from ventral cells (V(wt)) by microdissection. Discs and fragments were dissected in PBS and carefully washed several times to reduce the concentration of contaminating fat body cells.   RNA was extracted from imaginal disc fragments using the Mini RNA isolation kit (Zymo research). Linear RNA amplification was performed essentially as described (Klebes et al., 2002). In brief, total RNA was used for a first round of reverse transcription and in vitro transcription using T7 RNA polymerase. Subsequently, the amplified RNA product was subjected to a 2nd round of reverse transcription and in vitro transcription yielding 10,000-fold amplification or more.   Probes were indirectly labeled with amino-allyl modified dUTP in a reverse transcription reaction and coupled to reactive Cy3 or Cy5 dyes (Amersham). Correspondingly labeled Cy3 and Cy5 cDNA products were combined and purified on PCR purification columns (Qiagen), and diluted in hybridization buffer. Probes were added to the slides and incubated in a metal hybidization chamber (DieTech) in a waterbath at 65°C over night. Microarrays were washed and scanned on a GenePix 4000A scanner (Axon Instruments, Foster City, CA). Signal intensities were initially stored and analyzed using NOMAD (www.microarrays.org). Data was extracted as the global median normalized ratios (net median ratios) of the intensitites and log2 transformed. Signals with intensity values of the sum of median (foreground minus background) smaller than 300 were eliminated.  Each comparison was performed in replicates with independently isolated and processed probes. Within a set of replicate experiments, dyes were reversed to avoid bias in about half of the experiments. To represent all experiments with the same dye orientation the values of the log2 transformed expression ratios were reversed if necessary.

molecule: total RNA

organism: Drosophila melanogaster

source_name: Dorsal cells (D(wt))

tag: microdissected dorsal cells from prothoracic leg imaginal discs (D(wt)) isolated from wildtype larvae, 2 rounds of linear amplification

source_name: Ventral cells (V(wt))

tag: microdissected ventral cells from prothoracic leg imaginal discs (V(wt)) isolated from wildtype larvae, 2 rounds of linear amplification


title: Transdetermination study; D(wg) cells vs V(wg) cells_7-218

description: These experiments are described in Klebes et al. (Development, 2005, in press).  Comparisons – category #6,  slide 7-218,  V(wg) cells – probe 1 D(wg) cells – probe 2; (ratios are shown as probe1/probe2). Unique cell populations of Wingless over-expressing prothoracic leg imaginal discs of y,w,hs-flp; vgBE-GAL4/UASGFP, Actinc5, FRT y+, FRT, wg+ Drosophila larvae were compared with each other. In this experiment, dorsal cells (D(wg)) were compared to ventral cells (V(wg)) isolated from the same discs. These comparisons were intended to establish the unique expression profiles of dorsal and ventral leg imaginal cells in response to Wingless over-expression and serve as control experiments for the transdetermination study.  Prothoracic leg imaginal disc cells of the “weak point” that are competent to undergo transdetermination (TD cells) were labeled in vivo using the vgBE::GAL4/UAS-GFP reporter system (Sustar and Schubiger, 2005). Transdetermination was induced in these discs by over-expressing Wingless. TD cells were micro-dissected under a fluorescent dissecting scope. The remaining cells of the leg disc were separated into a dorsal (D(wg)) that lack the TD cells and ventral (V(wg)) fraction by cutting approximately along the median of the disc. Pooled fragments from 4-8 discs were used. Note that the D(wg) cell population represents all non-GFP-expressing dorsal cell of the leg disc. Ectopic Wg expression was induced by heat shock of young L3 larvae 60 h and 72h after egg deposition, and leg discs were isolated three days later. This protocol results in Wg over-expression in over 90% of the disc cells after heat shock (Maves and Schubiger, 1995). Note that Wg over-expression delays metamorphosis by one day, so these larvae had not yet pupariated. Discs and fragments were dissected in PBS and carefully washed several times to reduce the concentration of contaminating fat body cells.   RNA was extracted from imaginal disc fragments using the Mini RNA isolation kit (Zymo research). Linear RNA amplification was performed essentially as described (Klebes et al., 2002). In brief, total RNA was used for a first round of reverse transcription and in vitro transcription using T7 RNA polymerase. Subsequently, the amplified RNA product was subjected to a 2nd round of reverse transcription and in vitro transcription yielding 10,000-fold amplification or more.   Probes were indirectly labeled with amino-allyl modified dUTP in a reverse transcription reaction and coupled to reactive Cy3 or Cy5 dyes (Amersham). Correspondingly labeled Cy3 and Cy5 cDNA products were combined and purified on PCR purification columns (Qiagen), and diluted in hybridization buffer. Probes were added to the slides and incubated in a metal hybidization chamber (DieTech) in a waterbath at 65°C over night. Microarrays were washed and scanned on a GenePix 4000A scanner (Axon Instruments, Foster City, CA). Signal intensities were initially stored and analyzed using NOMAD (www.microarrays.org). Data was extracted as the global median normalized ratios (net median ratios) of the intensitites and log2 transformed. Signals with intensity values of the sum of median (foreground minus background) smaller than 300 were eliminated.  Each comparison was performed in replicates with independently isolated and processed probes. Within a set of replicate experiments, dyes were reversed to avoid bias in about half of the experiments. To represent all experiments with the same dye orientation the values of the log2 transformed expression ratios were reversed if necessary.

source_name: Dorsal (D(wg)) cells

tag: microdissected dorsal cells from Wingless over-expressing prothoracic leg imaginal discs (D(wg)), 2 rounds of linear amplification

source_name: Ventral (V(wg)) cells

tag: microdissected ventral cells from Wingless over-expressing prothoracic leg imaginal discs (V(wg)), 2 rounds of linear amplification


title: Transdetermination study; TD cells vs D(wt) cells_7-008

description: These experiments are described in Klebes et al. (Development, 2005, in press).  Comparisons – category #7,  slide 7-008, D(wt) cells – probe 1 vs. TD cells – probe 2 (ratios are shown as probe1/probe2).  The TD cell population of Wingless over-expressing prothoracic leg imaginal discs of y,w,hs-flp; vgBE-GAL4/UASGFP, Actinc5, FRT y+, FRT, wg+ Drosophila larvae was compared to dorsal cells (D(wt)) isolated from wildtype (OregonR) male third instar larvae. These comparisons were intended to establish the unique expression profiles of leg imaginal cells (TD cells) while they undergo a fate switch to transdetermine into wing cells.  Prothoracic leg imaginal disc cells of the “weak point” that are competent to undergo transdetermination (TD cells) were labeled in vivo using the vgBE::GAL4/UAS-GFP reporter system (Sustar and Schubiger, 2005). Transdetermination was induced in these discs by over-expressing Wingless. TD cells were micro-dissected under a fluorescent dissecting scope. Dorsal cells of wildtype prothoracic leg discs (D(wt)) were approximately separated from ventral cells (V(wt)) by microdissection.  Ectopic Wg expression was induced by heat shock of young L3 larvae 60 h and 72h after egg deposition, and leg discs were isolated three days later. This protocol results in Wg over-expression in over 90% of the disc cells after heat shock (Maves and Schubiger, 1995). Note that Wg over-expression delays metamorphosis by one day, so these larvae had not yet pupariated. Discs and fragments were dissected in PBS and carefully washed several times to reduce the concentration of contaminating fat body cells.   RNA was extracted from imaginal disc fragments using the Mini RNA isolation kit (Zymo research). Linear RNA amplification was performed essentially as described (Klebes et al., 2002). In brief, total RNA was used for a first round of reverse transcription and in vitro transcription using T7 RNA polymerase. Subsequently, the amplified RNA product was subjected to a 2nd round of reverse transcription and in vitro transcription yielding 10,000-fold amplification or more.   Probes were indirectly labeled with amino-allyl modified dUTP in a reverse transcription reaction and coupled to reactive Cy3 or Cy5 dyes (Amersham). Correspondingly labeled Cy3 and Cy5 cDNA products were combined and purified on PCR purification columns (Qiagen), and diluted in hybridization buffer. Probes were added to the slides and incubated in a metal hybidization chamber (DieTech) in a waterbath at 65°C over night. Microarrays were washed and scanned on a GenePix 4000A scanner (Axon Instruments, Foster City, CA). Signal intensities were initially stored and analyzed using NOMAD (www.microarrays.org). Data was extracted as the global median normalized ratios (net median ratios) of the intensitites and log2 transformed. Signals with intensity values of the sum of median (foreground minus background) smaller than 300 were eliminated.  Each comparison was performed in replicates with independently isolated and processed probes. Within a set of replicate experiments, dyes were reversed to avoid bias in about half of the experiments. To represent all experiments with the same dye orientation the values of the log2 transformed expression ratios were reversed if necessary.

source_name: Transdetermining (TD) cells

tag: microdissected TD cells from Wingless over-expressing prothoracic leg imaginal discs, 2 rounds of linear amplification

tag: microdissected dorsal cells from prothoracic leg imaginal discs D(wt) isolated from wildtype larvae, 2 rounds of linear amplification


title: Transdetermination study; regenerating discs vs reference_5-122

description: These experiments are described in Klebes et al. (Development, 2005, in press). Comparisons – category #9, slide 5-122, reference sample – probe 1 vs. regenerating disc – probe 2; (ratios are shown as probe1/probe2). In this experiment, regenerating disc cells were compared to the common leg disc reference sample. These comparisons were intended to establish the unique expression profiles of regenerating leg imaginal cells in response to fragmentation. Regenerating cells were obtained from disc fragments that had been cultured in the abdomen of female flies to promote proliferation. To minimize possible contamination with transdetermined cells, we did not use 1st leg discs, but instead examined the ‘3/4 fragment’ of 2nd leg discs (Sustar and Schubiger, 2005). This fragment regenerates missing parts, and in contrast to 1st leg discs, does not transdetermine (G.S., unpublished). The disc fragments were collected after 3-5 days of culture. The common reference sample was generated by pooling amplified RNA of male and female 1st, 2nd and 3rd leg imaginal discs in equal proportions. Discs and fragments were dissected in PBS and carefully washed several times to reduce the concentration of contaminating fat body cells. RNA was extracted from imaginal disc fragments using the Mini RNA isolation kit (Zymo research). Linear RNA amplification was performed essentially as described (Klebes et al., 2002). In brief, total RNA was used for a first round of reverse transcription and in vitro transcription using T7 RNA polymerase. Subsequently, the amplified RNA product was subjected to a 2nd round of reverse transcription and in vitro transcription yielding 10,000-fold amplification or more. Probes were indirectly labeled with amino-allyl modified dUTP in a reverse transcription reaction and coupled to reactive Cy3 or Cy5 dyes (Amersham). Correspondingly labeled Cy3 and Cy5 cDNA products were combined and purified on PCR purification columns (Qiagen), and diluted in hybridization buffer. Probes were added to the slides and incubated in a metal hybidization chamber (DieTech) in a waterbath at 65°C over night. Microarrays were washed and scanned on a GenePix 4000A scanner (Axon Instruments, Foster City, CA). Signal intensities were initially stored and analyzed using NOMAD (www.microarrays.org). Data was extracted as the global median normalized ratios (net median ratios) of the intensitites and log2 transformed. Signals with intensity values of the sum of median (foreground minus background) smaller than 300 were eliminated. Each comparison was performed in replicates with independently isolated and processed probes. Within a set of replicate experiments, dyes were reversed to avoid bias in about half of the experiments. To represent all experiments with the same dye orientation the values of the log2 transformed expression ratios were reversed if necessary.

source_name: Regenerating leg imaginal disc

tag: Cultivated 3/4 fragment of a mesothoracic (2nd) leg imaginal disc, 2 rounds of linear RNA amplification

source_name: Leg disc reference sample

tag: pooled amplified RNA of 1st, 2nd and 3rd leg discs of third instar male and female wildtype larvae


title: Transdetermination study; D(wt) cells vs V(wt) cells_7-018

description: These experiments are described in Klebes et al. (Development, 2005, in press).  Comparisons – category #8,  slide 7-018, D(wt), dorsal prothoracic leg disc cells (wildtype) – probe 1 vs. V(wt) ventral cells from the same discs– probe 2 (ratios are shown as probe 2/probe 1).  Dorsal cells (D(wt) of prothoracic leg imaginal discs were compared to ventral cells (V(wt)). Both cell populations were isolated from the same discs. For this experiment third instar larvae of Drosophila melanogaster strain OregonR (OR) were staged by feeding 1% bromphenol blue which allows to observe the gut content. Single male wandering late third instar larvae (half empty gut) were used to isolate leg discs. These comparisons were intended to establish the unique expression profiles of dorsal and ventral leg imaginal cells to distinguish them from genes identified in the transdeterminating cell population of Wingless over-expressing leg discs. Dorsal cells of wildtype prothoracic leg discs (D(wt)) were approximately separated from ventral cells (V(wt)) by microdissection. Discs and fragments were dissected in PBS and carefully washed several times to reduce the concentration of contaminating fat body cells.   RNA was extracted from imaginal disc fragments using the Mini RNA isolation kit (Zymo research). Linear RNA amplification was performed essentially as described (Klebes et al., 2002). In brief, total RNA was used for a first round of reverse transcription and in vitro transcription using T7 RNA polymerase. Subsequently, the amplified RNA product was subjected to a 2nd round of reverse transcription and in vitro transcription yielding 10,000-fold amplification or more.   Probes were indirectly labeled with amino-allyl modified dUTP in a reverse transcription reaction and coupled to reactive Cy3 or Cy5 dyes (Amersham). Correspondingly labeled Cy3 and Cy5 cDNA products were combined and purified on PCR purification columns (Qiagen), and diluted in hybridization buffer. Probes were added to the slides and incubated in a metal hybidization chamber (DieTech) in a waterbath at 65°C over night. Microarrays were washed and scanned on a GenePix 4000A scanner (Axon Instruments, Foster City, CA). Signal intensities were initially stored and analyzed using NOMAD (www.microarrays.org). Data was extracted as the global median normalized ratios (net median ratios) of the intensitites and log2 transformed. Signals with intensity values of the sum of median (foreground minus background) smaller than 300 were eliminated.  Each comparison was performed in replicates with independently isolated and processed probes. Within a set of replicate experiments, dyes were reversed to avoid bias in about half of the experiments. To represent all experiments with the same dye orientation the values of the log2 transformed expression ratios were reversed if necessary.

data_processing: log2 transformed global median normalized ratios (probe2/probe1)


title: Transdetermination study; regenerating discs vs reference_5-121

description: These experiments are described in Klebes et al. (Development, 2005, in press).  Comparisons – category #9,  slide 5-121, regenerating disc – probe 1 vs. reference sample – probe 2 (ratios are shown as probe2/probe1). In this experiment, regenerating disc cells were compared to the common leg disc reference sample. These comparisons were intended to establish the unique expression profiles of regenerating leg imaginal cells in response to fragmentation. Regenerating cells were obtained from disc fragments that had been cultured in the abdomen of female flies to promote proliferation. To minimize possible contamination with transdetermined cells, we did not use 1st leg discs, but instead examined the ‘3/4 fragment’ of 2nd leg discs (Sustar and Schubiger, 2005). This fragment regenerates missing parts, and in contrast to 1st leg discs, does not transdetermine (G.S., unpublished). The disc fragments were collected after 3-5 days of culture. The common reference sample was generated by pooling amplified RNA of male and female 1st, 2nd and 3rd leg imaginal discs in equal proportions. Discs and fragments were dissected in PBS and carefully washed several times to reduce the concentration of contaminating fat body cells.  RNA was extracted from imaginal disc fragments using the Mini RNA isolation kit (Zymo research). Linear RNA amplification was performed essentially as described (Klebes et al., 2002). In brief, total RNA was used for a first round of reverse transcription and in vitro transcription using T7 RNA polymerase. Subsequently, the amplified RNA product was subjected to a 2nd round of reverse transcription and in vitro transcription yielding 10,000-fold amplification or more. Probes were indirectly labeled with amino-allyl modified dUTP in a reverse transcription reaction and coupled to reactive Cy3 or Cy5 dyes (Amersham). Correspondingly labeled Cy3 and Cy5 cDNA products were combined and purified on PCR purification columns (Qiagen), and diluted in hybridization buffer. Probes were added to the slides and incubated in a metal hybidization chamber (DieTech) in a waterbath at 65°C over night. Microarrays were washed and scanned on a GenePix 4000A scanner (Axon Instruments, Foster City, CA). Signal intensities were initially stored and analyzed using NOMAD (www.microarrays.org). Data was extracted as the global median normalized ratios (net median ratios) of the intensitites and log2 transformed. Signals with intensity values of the sum of median (foreground minus background) smaller than 300 were eliminated.  Each comparison was performed in replicates with independently isolated and processed probes. Within a set of replicate experiments, dyes were reversed to avoid bias in about half of the experiments. To represent all experiments with the same dye orientation the values of the log2 transformed expression ratios were reversed if necessary.


title: Transdetermination study; TD cells vs D(wt) cells_7-017

description: These experiments are described in Klebes et al. (Development, 2005, in press).  Comparisons – category #7,  slide 7-017, D(wt) cells – probe 1 vs. TD cells – probe 2 (ratios are shown as probe1/probe2).  The TD cell population of Wingless over-expressing prothoracic leg imaginal discs of y,w,hs-flp; vgBE-GAL4/UASGFP, Actinc5, FRT y+, FRT, wg+ Drosophila larvae was compared to dorsal cells (D(wt)) isolated from wildtype (OregonR) male third instar larvae. These comparisons were intended to establish the unique expression profiles of leg imaginal cells (TD cells) while they undergo a fate switch to transdetermine into wing cells.  Prothoracic leg imaginal disc cells of the “weak point” that are competent to undergo transdetermination (TD cells) were labeled in vivo using the vgBE::GAL4/UAS-GFP reporter system (Sustar and Schubiger, 2005). Transdetermination was induced in these discs by over-expressing Wingless. TD cells were micro-dissected under a fluorescent dissecting scope. Dorsal cells of wildtype prothoracic leg discs (D(wt)) were approximately separated from ventral cells (V(wt)) by microdissection.  Ectopic Wg expression was induced by heat shock of young L3 larvae 60 h and 72h after egg deposition, and leg discs were isolated three days later. This protocol results in Wg over-expression in over 90% of the disc cells after heat shock (Maves and Schubiger, 1995). Note that Wg over-expression delays metamorphosis by one day, so these larvae had not yet pupariated. Discs and fragments were dissected in PBS and carefully washed several times to reduce the concentration of contaminating fat body cells.   RNA was extracted from imaginal disc fragments using the Mini RNA isolation kit (Zymo research). Linear RNA amplification was performed essentially as described (Klebes et al., 2002). In brief, total RNA was used for a first round of reverse transcription and in vitro transcription using T7 RNA polymerase. Subsequently, the amplified RNA product was subjected to a 2nd round of reverse transcription and in vitro transcription yielding 10,000-fold amplification or more.   Probes were indirectly labeled with amino-allyl modified dUTP in a reverse transcription reaction and coupled to reactive Cy3 or Cy5 dyes (Amersham). Correspondingly labeled Cy3 and Cy5 cDNA products were combined and purified on PCR purification columns (Qiagen), and diluted in hybridization buffer. Probes were added to the slides and incubated in a metal hybidization chamber (DieTech) in a waterbath at 65°C over night. Microarrays were washed and scanned on a GenePix 4000A scanner (Axon Instruments, Foster City, CA). Signal intensities were initially stored and analyzed using NOMAD (www.microarrays.org). Data was extracted as the global median normalized ratios (net median ratios) of the intensitites and log2 transformed. Signals with intensity values of the sum of median (foreground minus background) smaller than 300 were eliminated.  Each comparison was performed in replicates with independently isolated and processed probes. Within a set of replicate experiments, dyes were reversed to avoid bias in about half of the experiments. To represent all experiments with the same dye orientation the values of the log2 transformed expression ratios were reversed if necessary.


title: Transdetermination study; D(wg) cells vs V(wg) cells_7-046

description: These experiments are described in Klebes et al. (Development, 2005, in press).  Comparisons – category #6,  slide 7-046, D(wg) cells – probe 1 vs. V(wg) cells – probe 2; (ratios are shown as probe2/probe1). Unique cell populations of Wingless over-expressing prothoracic leg imaginal discs of y,w,hs-flp; vgBE-GAL4/UASGFP, Actinc5, FRT y+, FRT, wg+ Drosophila larvae were compared with each other. In this experiment, dorsal cells (D(wg)) were compared to ventral cells (V(wg)) isolated from the same discs. These comparisons were intended to establish the unique expression profiles of dorsal and ventral leg imaginal cells in response to Wingless over-expression and serve as control experiments for the transdetermination study.  Prothoracic leg imaginal disc cells of the “weak point” that are competent to undergo transdetermination (TD cells) were labeled in vivo using the vgBE::GAL4/UAS-GFP reporter system (Sustar and Schubiger, 2005). Transdetermination was induced in these discs by over-expressing Wingless. TD cells were micro-dissected under a fluorescent dissecting scope. The remaining cells of the leg disc were separated into a dorsal (D(wg)) that lack the TD cells and ventral (V(wg)) fraction by cutting approximately along the median of the disc. Pooled fragments from 4-8 discs were used. Note that the D(wg) cell population represents all non-GFP-expressing dorsal cell of the leg disc. Ectopic Wg expression was induced by heat shock of young L3 larvae 60 h and 72h after egg deposition, and leg discs were isolated three days later. This protocol results in Wg over-expression in over 90% of the disc cells after heat shock (Maves and Schubiger, 1995). Note that Wg over-expression delays metamorphosis by one day, so these larvae had not yet pupariated. Discs and fragments were dissected in PBS and carefully washed several times to reduce the concentration of contaminating fat body cells.   RNA was extracted from imaginal disc fragments using the Mini RNA isolation kit (Zymo research). Linear RNA amplification was performed essentially as described (Klebes et al., 2002). In brief, total RNA was used for a first round of reverse transcription and in vitro transcription using T7 RNA polymerase. Subsequently, the amplified RNA product was subjected to a 2nd round of reverse transcription and in vitro transcription yielding 10,000-fold amplification or more.   Probes were indirectly labeled with amino-allyl modified dUTP in a reverse transcription reaction and coupled to reactive Cy3 or Cy5 dyes (Amersham). Correspondingly labeled Cy3 and Cy5 cDNA products were combined and purified on PCR purification columns (Qiagen), and diluted in hybridization buffer. Probes were added to the slides and incubated in a metal hybidization chamber (DieTech) in a waterbath at 65°C over night. Microarrays were washed and scanned on a GenePix 4000A scanner (Axon Instruments, Foster City, CA). Signal intensities were initially stored and analyzed using NOMAD (www.microarrays.org). Data was extracted as the global median normalized ratios (net median ratios) of the intensitites and log2 transformed. Signals with intensity values of the sum of median (foreground minus background) smaller than 300 were eliminated.  Each comparison was performed in replicates with independently isolated and processed probes. Within a set of replicate experiments, dyes were reversed to avoid bias in about half of the experiments. To represent all experiments with the same dye orientation the values of the log2 transformed expression ratios were reversed if necessary.


title: Transdetermination study; TD cells vs V(wg) cells_7-035

description: These experiments are described in Klebes et al. (Development, 2005, in press).  Comparisons – category #5,  slide 7-035, TD cells– probe 1 vs. V(wg) cells – probe 2, (ratios are represented as probe2/probe1).  Unique cell populations of Wingless over-expressing prothoracic leg imaginal discs of y,w,hs-flp; vgBE-GAL4/UASGFP, Actinc5, FRT y+, FRT, wg+ Drosophila larvae were compared with each other. In this experiment, cells that undergo transdetermination (TD cells) were compared to ventral cells (V(wg)) isolated from the same discs. These comparisons were intended to establish the unique expression profiles of leg imaginal cells while they undergo a fate switch to transdetermine into wing cells.  Prothoracic leg imaginal disc cells of the “weak point” that are competent to undergo transdetermination (TD cells) were labeled in vivo using the vgBE::GAL4/UAS-GFP reporter system (Sustar and Schubiger, 2005). Transdetermination was induced in these discs by over-expressing Wingless. TD cells were micro-dissected under a fluorescent dissecting scope. The remaining cells of the leg disc were separated into a dorsal (D(wg)) that lack the TD cells and ventral (V(wg)) fraction by cutting approximately along the median of the disc. Pooled fragments from 4-8 discs were used  Ectopic Wg expression was induced by heat shock of young L3 larvae 60 h and 72h after egg deposition, and leg discs were isolated three days later. This protocol results in Wg over-expression in over 90% of the disc cells after heat shock (Maves and Schubiger, 1995). Note that Wg over-expression delays metamorphosis by one day, so these larvae had not yet pupariated. Discs and fragments were dissected in PBS and carefully washed several times to reduce the concentration of contaminating fat body cells.   RNA was extracted from imaginal disc fragments using the Mini RNA isolation kit (Zymo research). Linear RNA amplification was performed essentially as described (Klebes et al., 2002). In brief, total RNA was used for a first round of reverse transcription and in vitro transcription using T7 RNA polymerase. Subsequently, the amplified RNA product was subjected to a 2nd round of reverse transcription and in vitro transcription yielding 10,000-fold amplification or more.   Probes were indirectly labeled with amino-allyl modified dUTP in a reverse transcription reaction and coupled to reactive Cy3 or Cy5 dyes (Amersham). Correspondingly labeled Cy3 and Cy5 cDNA products were combined and purified on PCR purification columns (Qiagen), and diluted in hybridization buffer. Probes were added to the slides and incubated in a metal hybidization chamber (DieTech) in a waterbath at 65°C over night. Microarrays were washed and scanned on a GenePix 4000A scanner (Axon Instruments, Foster City, CA). Signal intensities were initially stored and analyzed using NOMAD (www.microarrays.org). Data was extracted as the global median normalized ratios (net median ratios) of the intensitites and log2 transformed. Signals with intensity values of the sum of median (foreground minus background) smaller than 300 were eliminated.  Each comparison was performed in replicates with independently isolated and processed probes. Within a set of replicate experiments, dyes were reversed to avoid bias in about half of the experiments. To represent all experiments with the same dye orientation the values of the log2 transformed expression ratios were reversed if necessary.


title: Transdetermination study; TD cells vs V(wg) cells_7-044

description: These experiments are described in Klebes et al. (Development, 2005, in press).  Comparisons – category #5,  slide 7-044, V(wg) cells – probe 1 vs. TD cells– probe 2 (ratios are represented as probe1/probe2).  Unique cell populations of Wingless over-expressing prothoracic leg imaginal discs of y,w,hs-flp; vgBE-GAL4/UASGFP, Actinc5, FRT y+, FRT, wg+ Drosophila larvae were compared with each other. In this experiment, cells that undergo transdetermination (TD cells) were compared to ventral cells (V(wg)) isolated from the same discs. These comparisons were intended to establish the unique expression profiles of leg imaginal cells while they undergo a fate switch to transdetermine into wing cells.  Prothoracic leg imaginal disc cells of the “weak point” that are competent to undergo transdetermination (TD cells) were labeled in vivo using the vgBE::GAL4/UAS-GFP reporter system (Sustar and Schubiger, 2005). Transdetermination was induced in these discs by over-expressing Wingless. TD cells were micro-dissected under a fluorescent dissecting scope. The remaining cells of the leg disc were separated into a dorsal (D(wg)) that lack the TD cells and ventral (V(wg)) fraction by cutting approximately along the median of the disc. Pooled fragments from 4-8 discs were used  Ectopic Wg expression was induced by heat shock of young L3 larvae 60 h and 72h after egg deposition, and leg discs were isolated three days later. This protocol results in Wg over-expression in over 90% of the disc cells after heat shock (Maves and Schubiger, 1995). Note that Wg over-expression delays metamorphosis by one day, so these larvae had not yet pupariated. Discs and fragments were dissected in PBS and carefully washed several times to reduce the concentration of contaminating fat body cells.   RNA was extracted from imaginal disc fragments using the Mini RNA isolation kit (Zymo research). Linear RNA amplification was performed essentially as described (Klebes et al., 2002). In brief, total RNA was used for a first round of reverse transcription and in vitro transcription using T7 RNA polymerase. Subsequently, the amplified RNA product was subjected to a 2nd round of reverse transcription and in vitro transcription yielding 10,000-fold amplification or more.   Probes were indirectly labeled with amino-allyl modified dUTP in a reverse transcription reaction and coupled to reactive Cy3 or Cy5 dyes (Amersham). Correspondingly labeled Cy3 and Cy5 cDNA products were combined and purified on PCR purification columns (Qiagen), and diluted in hybridization buffer. Probes were added to the slides and incubated in a metal hybidization chamber (DieTech) in a waterbath at 65°C over night. Microarrays were washed and scanned on a GenePix 4000A scanner (Axon Instruments, Foster City, CA). Signal intensities were initially stored and analyzed using NOMAD (www.microarrays.org). Data was extracted as the global median normalized ratios (net median ratios) of the intensitites and log2 transformed. Signals with intensity values of the sum of median (foreground minus background) smaller than 300 were eliminated.  Each comparison was performed in replicates with independently isolated and processed probes. Within a set of replicate experiments, dyes were reversed to avoid bias in about half of the experiments. To represent all experiments with the same dye orientation the values of the log2 transformed expression ratios were reversed if necessary.


title: Transdetermination study; TD cells vs D(wg) cells_7-041

description: These experiments are described in Klebes et al. (Development, 2005, in press).  Comparisons – category #4,  slide 7-041, TD cells – probe 1 vs. D(wg) cells – probe 2, (ratios are shown as probe2/probe1).  Unique cell populations of Wingless over-expressing prothoracic leg imaginal discs of y,w,hs-flp; vgBE-GAL4/UASGFP, Actinc5, FRT y+, FRT, wg+ Drosophila larvae were compared with each other. In this experiment, cells that undergo transdetermination (TD cells) were compared to dorsal cells (D(wg)) isolated from the same discs. These comparisons were intended to establish the unique expression profiles of leg imaginal cells (TD cells) while they undergo a fate switch to transdetermine into wing cells.  Prothoracic leg imaginal disc cells of the “weak point” that are competent to undergo transdetermination (TD cells) were labeled in vivo using the vgBE::GAL4/UAS-GFP reporter system (Sustar and Schubiger, 2005). Transdetermination was induced in these discs by over-expressing Wingless. TD cells were micro-dissected under a fluorescent dissecting scope. The remaining cells of the leg disc were separated into a dorsal (D(wg)) that lack the TD cells and ventral (V(wg)) fraction by cutting approximately along the median of the disc. Pooled fragments from 4-8 discs were used. Note that the D(wg) cell population represents all non-GFP-expressing dorsal cell of the leg disc.  Ectopic Wg expression was induced by heat shock of young L3 larvae 60 h and 72h after egg deposition, and leg discs were isolated three days later. This protocol results in Wg over-expression in over 90% of the disc cells after heat shock (Maves and Schubiger, 1995). Note that Wg over-expression delays metamorphosis by one day, so these larvae had not yet pupariated. Discs and fragments were dissected in PBS and carefully washed several times to reduce the concentration of contaminating fat body cells.   RNA was extracted from imaginal disc fragments using the Mini RNA isolation kit (Zymo research). Linear RNA amplification was performed essentially as described (Klebes et al., 2002). In brief, total RNA was used for a first round of reverse transcription and in vitro transcription using T7 RNA polymerase. Subsequently, the amplified RNA product was subjected to a 2nd round of reverse transcription and in vitro transcription yielding 10,000-fold amplification or more.   Probes were indirectly labeled with amino-allyl modified UTP in a reverse transcription reaction and coupled to reactive Cy3 or Cy5 dyes (Amersham). Correspondingly labeled Cy3 and Cy5 cDNA products were combined and purified on PCR purification columns (Qiagen), and diluted in hybridization buffer. Probes were added to the slides and incubated in a metal hybidization chamber (DieTech) in a waterbath at 65°C over night. Microarrays were washed and scanned on a GenePix 4000A scanner (Axon Instruments, Foster City, CA). Signal intensities were initially stored and analyzed using NOMAD (www.microarrays.org). Data was extracted as the global median normalized ratios (net median ratios) of the intensitites and log2 transformed. Signals with intensity values of the sum of median (foreground minus background) smaller than 300 were eliminated.  Each comparison was performed in replicates with independently isolated and processed probes. Within a set of replicate experiments, dyes were reversed to avoid bias in about half of the experiments. To represent all experiments with the same dye orientation the values of the log2 transformed expression ratios were reversed if necessary.

tag: microdissected dorsal cells from Wingless over-expressing prothoracic leg imaginal discs D(wg), 2 rounds of linear amplification


title: Transdetermination study; TD cells vs D(wg) cells_7-047

description: These experiments are described in Klebes et al. (Development, 2005, in press).  Comparisons – category #4,  slide 7-047,  D(wg) cells – probe 1 VS. TD cells – probe 2, (ratios are shown as probe1/probe2).  Unique cell populations of Wingless over-expressing prothoracic leg imaginal discs of y,w,hs-flp; vgBE-GAL4/UASGFP, Actinc5, FRT y+, FRT, wg+ Drosophila larvae were compared with each other. In this experiment, cells that undergo transdetermination (TD cells) were compared to dorsal cells (D(wg)) isolated from the same discs. These comparisons were intended to establish the unique expression profiles of leg imaginal cells (TD cells) while they undergo a fate switch to transdetermine into wing cells.  Prothoracic leg imaginal disc cells of the “weak point” that are competent to undergo transdetermination (TD cells) were labeled in vivo using the vgBE::GAL4/UAS-GFP reporter system (Sustar and Schubiger, 2005). Transdetermination was induced in these discs by over-expressing Wingless. TD cells were micro-dissected under a fluorescent dissecting scope. The remaining cells of the leg disc were separated into a dorsal (D(wg)) that lack the TD cells and ventral (V(wg)) fraction by cutting approximately along the median of the disc. Pooled fragments from 4-8 discs were used. Note that the D(wg) cell population represents all non-GFP-expressing dorsal cell of the leg disc.  Ectopic Wg expression was induced by heat shock of young L3 larvae 60 h and 72h after egg deposition, and leg discs were isolated three days later. This protocol results in Wg over-expression in over 90% of the disc cells after heat shock (Maves and Schubiger, 1995). Note that Wg over-expression delays metamorphosis by one day, so these larvae had not yet pupariated. Discs and fragments were dissected in PBS and carefully washed several times to reduce the concentration of contaminating fat body cells.   RNA was extracted from imaginal disc fragments using the Mini RNA isolation kit (Zymo research). Linear RNA amplification was performed essentially as described (Klebes et al., 2002). In brief, total RNA was used for a first round of reverse transcription and in vitro transcription using T7 RNA polymerase. Subsequently, the amplified RNA product was subjected to a 2nd round of reverse transcription and in vitro transcription yielding 10,000-fold amplification or more.   Probes were indirectly labeled with amino-allyl modified UTP in a reverse transcription reaction and coupled to reactive Cy3 or Cy5 dyes (Amersham). Correspondingly labeled Cy3 and Cy5 cDNA products were combined and purified on PCR purification columns (Qiagen), and diluted in hybridization buffer. Probes were added to the slides and incubated in a metal hybidization chamber (DieTech) in a waterbath at 65°C over night. Microarrays were washed and scanned on a GenePix 4000A scanner (Axon Instruments, Foster City, CA). Signal intensities were initially stored and analyzed using NOMAD (www.microarrays.org). Data was extracted as the global median normalized ratios (net median ratios) of the intensitites and log2 transformed. Signals with intensity values of the sum of median (foreground minus background) smaller than 300 were eliminated.  Each comparison was performed in replicates with independently isolated and processed probes. Within a set of replicate experiments, dyes were reversed to avoid bias in about half of the experiments. To represent all experiments with the same dye orientation the values of the log2 transformed expression ratios were reversed if necessary.


title: Transdetermination study; TD cells vs V(wg) cells_7-050

description: These experiments are described in Klebes et al. (Development, 2005, in press).  Comparisons – category #5,  slide 7-050, TD cells– probe 1 vs. V(wg) cells – probe 2, (ratios are represented as probe2/probe1).  Unique cell populations of Wingless over-expressing prothoracic leg imaginal discs of y,w,hs-flp; vgBE-GAL4/UASGFP, Actinc5, FRT y+, FRT, wg+ Drosophila larvae were compared with each other. In this experiment, cells that undergo transdetermination (TD cells) were compared to ventral cells (V(wg)) isolated from the same discs. These comparisons were intended to establish the unique expression profiles of leg imaginal cells while they undergo a fate switch to transdetermine into wing cells.  Prothoracic leg imaginal disc cells of the “weak point” that are competent to undergo transdetermination (TD cells) were labeled in vivo using the vgBE::GAL4/UAS-GFP reporter system (Sustar and Schubiger, 2005). Transdetermination was induced in these discs by over-expressing Wingless. TD cells were micro-dissected under a fluorescent dissecting scope. The remaining cells of the leg disc were separated into a dorsal (D(wg)) that lack the TD cells and ventral (V(wg)) fraction by cutting approximately along the median of the disc. Pooled fragments from 4-8 discs were used  Ectopic Wg expression was induced by heat shock of young L3 larvae 60 h and 72h after egg deposition, and leg discs were isolated three days later. This protocol results in Wg over-expression in over 90% of the disc cells after heat shock (Maves and Schubiger, 1995). Note that Wg over-expression delays metamorphosis by one day, so these larvae had not yet pupariated. Discs and fragments were dissected in PBS and carefully washed several times to reduce the concentration of contaminating fat body cells.   RNA was extracted from imaginal disc fragments using the Mini RNA isolation kit (Zymo research). Linear RNA amplification was performed essentially as described (Klebes et al., 2002). In brief, total RNA was used for a first round of reverse transcription and in vitro transcription using T7 RNA polymerase. Subsequently, the amplified RNA product was subjected to a 2nd round of reverse transcription and in vitro transcription yielding 10,000-fold amplification or more.   Probes were indirectly labeled with amino-allyl modified dUTP in a reverse transcription reaction and coupled to reactive Cy3 or Cy5 dyes (Amersham). Correspondingly labeled Cy3 and Cy5 cDNA products were combined and purified on PCR purification columns (Qiagen), and diluted in hybridization buffer. Probes were added to the slides and incubated in a metal hybidization chamber (DieTech) in a waterbath at 65°C over night. Microarrays were washed and scanned on a GenePix 4000A scanner (Axon Instruments, Foster City, CA). Signal intensities were initially stored and analyzed using NOMAD (www.microarrays.org). Data was extracted as the global median normalized ratios (net median ratios) of the intensitites and log2 transformed. Signals with intensity values of the sum of median (foreground minus background) smaller than 300 were eliminated.  Each comparison was performed in replicates with independently isolated and processed probes. Within a set of replicate experiments, dyes were reversed to avoid bias in about half of the experiments. To represent all experiments with the same dye orientation the values of the log2 transformed expression ratios were reversed if necessary.


title: Transdetermination study; TD cells vs V(wg) cells_7-226

description: These experiments are described in Klebes et al. (Development, 2005, in press).  Comparisons – category #5,  slide 7-226, TD cells– probe 1 vs. V(wg) cells – probe 2, (ratios are represented as probe2/probe1).  Unique cell populations of Wingless over-expressing prothoracic leg imaginal discs of y,w,hs-flp; vgBE-GAL4/UASGFP, Actinc5, FRT y+, FRT, wg+ Drosophila larvae were compared with each other. In this experiment, cells that undergo transdetermination (TD cells) were compared to ventral cells (V(wg)) isolated from the same discs. These comparisons were intended to establish the unique expression profiles of leg imaginal cells while they undergo a fate switch to transdetermine into wing cells.  Prothoracic leg imaginal disc cells of the “weak point” that are competent to undergo transdetermination (TD cells) were labeled in vivo using the vgBE::GAL4/UAS-GFP reporter system (Sustar and Schubiger, 2005). Transdetermination was induced in these discs by over-expressing Wingless. TD cells were micro-dissected under a fluorescent dissecting scope. The remaining cells of the leg disc were separated into a dorsal (D(wg)) that lack the TD cells and ventral (V(wg)) fraction by cutting approximately along the median of the disc. Pooled fragments from 4-8 discs were used  Ectopic Wg expression was induced by heat shock of young L3 larvae 60 h and 72h after egg deposition, and leg discs were isolated three days later. This protocol results in Wg over-expression in over 90% of the disc cells after heat shock (Maves and Schubiger, 1995). Note that Wg over-expression delays metamorphosis by one day, so these larvae had not yet pupariated. Discs and fragments were dissected in PBS and carefully washed several times to reduce the concentration of contaminating fat body cells.   RNA was extracted from imaginal disc fragments using the Mini RNA isolation kit (Zymo research). Linear RNA amplification was performed essentially as described (Klebes et al., 2002). In brief, total RNA was used for a first round of reverse transcription and in vitro transcription using T7 RNA polymerase. Subsequently, the amplified RNA product was subjected to a 2nd round of reverse transcription and in vitro transcription yielding 10,000-fold amplification or more.   Probes were indirectly labeled with amino-allyl modified dUTP in a reverse transcription reaction and coupled to reactive Cy3 or Cy5 dyes (Amersham). Correspondingly labeled Cy3 and Cy5 cDNA products were combined and purified on PCR purification columns (Qiagen), and diluted in hybridization buffer. Probes were added to the slides and incubated in a metal hybidization chamber (DieTech) in a waterbath at 65°C over night. Microarrays were washed and scanned on a GenePix 4000A scanner (Axon Instruments, Foster City, CA). Signal intensities were initially stored and analyzed using NOMAD (www.microarrays.org). Data was extracted as the global median normalized ratios (net median ratios) of the intensitites and log2 transformed. Signals with intensity values of the sum of median (foreground minus background) smaller than 300 were eliminated.  Each comparison was performed in replicates with independently isolated and processed probes. Within a set of replicate experiments, dyes were reversed to avoid bias in about half of the experiments. To represent all experiments with the same dye orientation the values of the log2 transformed expression ratios were reversed if necessary.


title: Transdetermination study; wg-induced 1st leg disc vs reference_7-067

description: These experiments are described in Klebes et al. (Development, 2005, in press).  Comparisons – category #3,  slide 7-067, reference sample - probe 1 vs. Wingless over-expressing prothoracic leg disc – probe 2.  (ratios are shown as probe1/probe2). Leg imaginal discs of single y,w,hs-flp; vgBE-GAL4/UASGFP, Actinc5, FRT y+, FRT, wg+ Drosophila larvae were compared to a common reference sample. These comparisons were intended to establish the unique expression profiles of Wingless over-expressing leg imaginal discs that undergo transdetermination to form wing tissue. Ectopic Wg expression was induced by heat shock of young L3 larvae 60 h and 72h after egg deposition, and leg discs were isolated three days later. This protocol results in Wg over-expression in over 90% of the disc cells after heat shock (Maves and Schubiger, 1995).  Note that Wg over-expression delays metamorphosis by one day, so these larvae had not yet pupariated. Discs were dissected in PBS and carefully washed several times to reduce the concentration of contaminating fat body cells.   RNA was extracted from single imaginal discs using the Mini RNA isolation kit (Zymo research). Linear RNA amplification was performed essentially as described (Klebes et al., 2002). In brief, total RNA was used for a first round of reverse transcription and in vitro transcription using T7 RNA polymerase. Subsequently, the amplified RNA product was subjected to a 2nd round of reverse transcription and in vitro transcription yielding 10,000-fold amplification or more.   Probes were indirectly labeled with amino-allyl modified dUTP in a reverse transcription reaction and coupled to reactive Cy3 or Cy5 dyes (Amersham). Correspondingly labeled Cy3 and Cy5 cDNA products were combined and purified on PCR purification columns (Qiagen), and diluted in hybridization buffer. Probes were added to the slides and incubated in a metal hybidization chamber (DieTech) in a waterbath at 65°C over night. Microarrays were washed and scanned on a GenePix 4000A scanner (Axon Instruments, Foster City, CA). Signal intensities were initially stored and analyzed using NOMAD (www.microarrays.org). Data was extracted as the global median normalized ratios (net median ratios) of the intensitites and log2 transformed. Signals with intensity values of the sum of median (foreground minus background) smaller than 300 were eliminated.  Each comparison was performed in replicates with independently isolated and processed probes. Within a set of replicate experiments, dyes were reversed to avoid bias in about half of the experiments. To represent all experiments with the same dye orientation the values of the log2 transformed expression ratios were reversed if necessary.

source_name: wingless over-expressing prothoracic leg imaginal disc

tag: 2 rounds of linear RNA amplification on Wingless over-expressing prothoracic leg imaginal disc


title: Transdetermination study; wg-induced 1st leg disc vs reference_7-078

description: These experiments are described in Klebes et al. (Development, 2005, in press).  Comparisons – category #3,  slide 7-078, reference sample - probe 1 vs. Wingless over-expressing prothoracic leg disc – probe 2.  (ratios are shown as probe1/probe2). Leg imaginal discs of single y,w,hs-flp; vgBE-GAL4/UASGFP, Actinc5, FRT y+, FRT, wg+ Drosophila larvae were compared to a common reference sample. These comparisons were intended to establish the unique expression profiles of Wingless over-expressing leg imaginal discs that undergo transdetermination to form wing tissue. Ectopic Wg expression was induced by heat shock of young L3 larvae 60 h and 72h after egg deposition, and leg discs were isolated three days later. This protocol results in Wg over-expression in over 90% of the disc cells after heat shock (Maves and Schubiger, 1995).  Note that Wg over-expression delays metamorphosis by one day, so these larvae had not yet pupariated. Discs were dissected in PBS and carefully washed several times to reduce the concentration of contaminating fat body cells.   RNA was extracted from single imaginal discs using the Mini RNA isolation kit (Zymo research). Linear RNA amplification was performed essentially as described (Klebes et al., 2002). In brief, total RNA was used for a first round of reverse transcription and in vitro transcription using T7 RNA polymerase. Subsequently, the amplified RNA product was subjected to a 2nd round of reverse transcription and in vitro transcription yielding 10,000-fold amplification or more.   Probes were indirectly labeled with amino-allyl modified dUTP in a reverse transcription reaction and coupled to reactive Cy3 or Cy5 dyes (Amersham). Correspondingly labeled Cy3 and Cy5 cDNA products were combined and purified on PCR purification columns (Qiagen), and diluted in hybridization buffer. Probes were added to the slides and incubated in a metal hybidization chamber (DieTech) in a waterbath at 65°C over night. Microarrays were washed and scanned on a GenePix 4000A scanner (Axon Instruments, Foster City, CA). Signal intensities were initially stored and analyzed using NOMAD (www.microarrays.org). Data was extracted as the global median normalized ratios (net median ratios) of the intensitites and log2 transformed. Signals with intensity values of the sum of median (foreground minus background) smaller than 300 were eliminated.  Each comparison was performed in replicates with independently isolated and processed probes. Within a set of replicate experiments, dyes were reversed to avoid bias in about half of the experiments. To represent all experiments with the same dye orientation the values of the log2 transformed expression ratios were reversed if necessary.


title: Transdetermination study; wg-induced 1st leg disc vs reference_7-065

description: These experiments are described in Klebes et al. (Development, 2005, in press).  Comparisons – category #3,  slide 7-065, Wingless over-expressing prothoracic leg disc – probe 1 vs. reference sample – probe 2.  (ratios are shown as probe2/probe1). Leg imaginal discs of single y,w,hs-flp; vgBE-GAL4/UASGFP, Actinc5, FRT y+, FRT, wg+ Drosophila larvae were compared to a common reference sample. These comparisons were intended to establish the unique expression profiles of Wingless over-expressing leg imaginal discs that undergo transdetermination to form wing tissue. Ectopic Wg expression was induced by heat shock of young L3 larvae 60 h and 72h after egg deposition, and leg discs were isolated three days later. This protocol results in Wg over-expression in over 90% of the disc cells after heat shock (Maves and Schubiger, 1995).  Note that Wg over-expression delays metamorphosis by one day, so these larvae had not yet pupariated. Discs were dissected in PBS and carefully washed several times to reduce the concentration of contaminating fat body cells.   RNA was extracted from single imaginal discs using the Mini RNA isolation kit (Zymo research). Linear RNA amplification was performed essentially as described (Klebes et al., 2002). In brief, total RNA was used for a first round of reverse transcription and in vitro transcription using T7 RNA polymerase. Subsequently, the amplified RNA product was subjected to a 2nd round of reverse transcription and in vitro transcription yielding 10,000-fold amplification or more.   Probes were indirectly labeled with amino-allyl modified dUTP in a reverse transcription reaction and coupled to reactive Cy3 or Cy5 dyes (Amersham). Correspondingly labeled Cy3 and Cy5 cDNA products were combined and purified on PCR purification columns (Qiagen), and diluted in hybridization buffer. Probes were added to the slides and incubated in a metal hybidization chamber (DieTech) in a waterbath at 65°C over night. Microarrays were washed and scanned on a GenePix 4000A scanner (Axon Instruments, Foster City, CA). Signal intensities were initially stored and analyzed using NOMAD (www.microarrays.org). Data was extracted as the global median normalized ratios (net median ratios) of the intensitites and log2 transformed. Signals with intensity values of the sum of median (foreground minus background) smaller than 300 were eliminated.  Each comparison was performed in replicates with independently isolated and processed probes. Within a set of replicate experiments, dyes were reversed to avoid bias in about half of the experiments. To represent all experiments with the same dye orientation the values of the log2 transformed expression ratios were reversed if necessary.


title: Transdetermination study; wg-induced 1st leg disc vs reference_6-069

description: These experiments are described in Klebes et al. (Development, 2005, in press).  Comparisons – category #3,  slide 6-069, reference sample - probe 1 vs. Wingless over-expressing prothoracic leg disc – probe 2.  (ratios are shown as probe1/probe2). Leg imaginal discs of single y,w,hs-flp; vgBE-GAL4/UASGFP, Actinc5, FRT y+, FRT, wg+ Drosophila larvae were compared to a common reference sample. These comparisons were intended to establish the unique expression profiles of Wingless over-expressing leg imaginal discs that undergo transdetermination to form wing tissue. Ectopic Wg expression was induced by heat shock of young L3 larvae 60 h and 72h after egg deposition, and leg discs were isolated three days later. This protocol results in Wg over-expression in over 90% of the disc cells after heat shock (Maves and Schubiger, 1995).  Note that Wg over-expression delays metamorphosis by one day, so these larvae had not yet pupariated. Discs were dissected in PBS and carefully washed several times to reduce the concentration of contaminating fat body cells.   RNA was extracted from single imaginal discs using the Mini RNA isolation kit (Zymo research). Linear RNA amplification was performed essentially as described (Klebes et al., 2002). In brief, total RNA was used for a first round of reverse transcription and in vitro transcription using T7 RNA polymerase. Subsequently, the amplified RNA product was subjected to a 2nd round of reverse transcription and in vitro transcription yielding 10,000-fold amplification or more.   Probes were indirectly labeled with amino-allyl modified dUTP in a reverse transcription reaction and coupled to reactive Cy3 or Cy5 dyes (Amersham). Correspondingly labeled Cy3 and Cy5 cDNA products were combined and purified on PCR purification columns (Qiagen), and diluted in hybridization buffer. Probes were added to the slides and incubated in a metal hybidization chamber (DieTech) in a waterbath at 65°C over night. Microarrays were washed and scanned on a GenePix 4000A scanner (Axon Instruments, Foster City, CA). Signal intensities were initially stored and analyzed using NOMAD (www.microarrays.org). Data was extracted as the global median normalized ratios (net median ratios) of the intensitites and log2 transformed. Signals with intensity values of the sum of median (foreground minus background) smaller than 300 were eliminated.  Each comparison was performed in replicates with independently isolated and processed probes. Within a set of replicate experiments, dyes were reversed to avoid bias in about half of the experiments. To represent all experiments with the same dye orientation the values of the log2 transformed expression ratios were reversed if necessary.


title: Transdetermination study; wg-induced 1st leg disc vs reference_7-049

description: These experiments are described in Klebes et al. (Development, 2005, in press).  Comparisons – category #3,  slide 7-049, Wingless over-expressing prothoracic leg disc – probe 1 vs. reference sample – probe 2.  (ratios are shown as probe2/probe1). Leg imaginal discs of single y,w,hs-flp; vgBE-GAL4/UASGFP, Actinc5, FRT y+, FRT, wg+ Drosophila larvae were compared to a common reference sample. These comparisons were intended to establish the unique expression profiles of Wingless over-expressing leg imaginal discs that undergo transdetermination to form wing tissue. Ectopic Wg expression was induced by heat shock of young L3 larvae 60 h and 72h after egg deposition, and leg discs were isolated three days later. This protocol results in Wg over-expression in over 90% of the disc cells after heat shock (Maves and Schubiger, 1995).  Note that Wg over-expression delays metamorphosis by one day, so these larvae had not yet pupariated. Discs were dissected in PBS and carefully washed several times to reduce the concentration of contaminating fat body cells.   RNA was extracted from single imaginal discs using the Mini RNA isolation kit (Zymo research). Linear RNA amplification was performed essentially as described (Klebes et al., 2002). In brief, total RNA was used for a first round of reverse transcription and in vitro transcription using T7 RNA polymerase. Subsequently, the amplified RNA product was subjected to a 2nd round of reverse transcription and in vitro transcription yielding 10,000-fold amplification or more.   Probes were indirectly labeled with amino-allyl modified dUTP in a reverse transcription reaction and coupled to reactive Cy3 or Cy5 dyes (Amersham). Correspondingly labeled Cy3 and Cy5 cDNA products were combined and purified on PCR purification columns (Qiagen), and diluted in hybridization buffer. Probes were added to the slides and incubated in a metal hybidization chamber (DieTech) in a waterbath at 65°C over night. Microarrays were washed and scanned on a GenePix 4000A scanner (Axon Instruments, Foster City, CA). Signal intensities were initially stored and analyzed using NOMAD (www.microarrays.org). Data was extracted as the global median normalized ratios (net median ratios) of the intensitites and log2 transformed. Signals with intensity values of the sum of median (foreground minus background) smaller than 300 were eliminated.  Each comparison was performed in replicates with independently isolated and processed probes. Within a set of replicate experiments, dyes were reversed to avoid bias in about half of the experiments. To represent all experiments with the same dye orientation the values of the log2 transformed expression ratios were reversed if necessary.


title: Transdetermination study; wing vs leg_5-254

description: These experiments are described in Klebes et al. (Development, 2005, in press). Comparisons – category #2, slide 5-254, metathoracic leg disc – probe 1 vs. wing disc – probe 2. (ratios are shown as probe1/probe2). Wing imaginal discs of single wildtype Drosophila larvae were compared to a single leg imaginal disc of the same animal. In this experiment the leg disc was a metathoracic (3rd) leg imaginal disc. These comparisons were intended to establish the unique expression profiles of wing and leg imaginal discs. For this experiment wing and leg imaginal discs of third instar larvae of Drosophila melanogaster strain OregonR (OR) were staged by feeding 1% bromphenol blue which allows to observe the gut content. Single male wandering late third instar larvae (half empty gut) were used to isolate wing and leg discs. Discs were dissected in PBS and carefully washed several times to reduce the concentration of contaminating fat body cells. RNA was extracted from single imaginal discs using the Mini RNA isolation kit (Zymo research). Linear RNA amplification was performed essentially as described (Klebes et al., 2002). In brief, total RNA was used for a first round of reverse transcription and in vitro transcription using T7 RNA polymerase. Subsequently, the amplified RNA product was subjected to a 2nd round of reverse transcription and in vitro transcription yielding 10,000-fold amplification or more. Probes were indirectly labeled with amino-allyl modified dUTP in a reverse transcription reaction and coupled to reactive Cy3 or Cy5 dyes (Amersham). Correspondingly labeled Cy3 and Cy5 cDNA products were combined and purified on PCR purification columns (Qiagen), and diluted in hybridization buffer. Probes were added to the slides and incubated in a metal hybidization chamber (DieTech) in a waterbath at 65°C over night. Microarrays were washed and scanned on a GenePix 4000A scanner (Axon Instruments, Foster City, CA). Signal intensities were initially stored and analyzed using NOMAD (www.microarrays.org). Data was extracted as the global median normalized ratios (net median ratios) of the intensitites and log2 transformed. Signals with intensity values of the sum of median (foreground minus background) smaller than 300 were eliminated. Each comparison was performed in replicates with independently isolated and processed probes. Within a set of replicate experiments, dyes were reversed to avoid bias in about half of the experiments. To represent all experiments with the same dye orientation the values of the log2 transformed expression ratios were reversed if necessary.

source_name: wing imaginal disc

tag: wildtype (OR) wing imaginal disc of third instar male larva

source_name: metathoracic leg imaginal disc

tag: wildtype (OR) metathoracic leg imaginal disc of third instar male larva


title: Transdetermination study; wg-induced 1st leg disc vs reference_7-159

description: These experiments are described in Klebes et al. (Development, 2005, in press).  Comparisons – category #3,  slide 7-159, Wingless over-expressing prothoracic leg disc – probe 1 vs. reference sample – probe 2.  (ratios are shown as probe2/probe1). Leg imaginal discs of single y,w,hs-flp; vgBE-GAL4/UASGFP, Actinc5, FRT y+, FRT, wg+ Drosophila larvae were compared to a common reference sample. These comparisons were intended to establish the unique expression profiles of Wingless over-expressing leg imaginal discs that undergo transdetermination to form wing tissue. Ectopic Wg expression was induced by heat shock of young L3 larvae 60 h and 72h after egg deposition, and leg discs were isolated three days later. This protocol results in Wg over-expression in over 90% of the disc cells after heat shock (Maves and Schubiger, 1995).  Note that Wg over-expression delays metamorphosis by one day, so these larvae had not yet pupariated. Discs were dissected in PBS and carefully washed several times to reduce the concentration of contaminating fat body cells.   RNA was extracted from single imaginal discs using the Mini RNA isolation kit (Zymo research). Linear RNA amplification was performed essentially as described (Klebes et al., 2002). In brief, total RNA was used for a first round of reverse transcription and in vitro transcription using T7 RNA polymerase. Subsequently, the amplified RNA product was subjected to a 2nd round of reverse transcription and in vitro transcription yielding 10,000-fold amplification or more.   Probes were indirectly labeled with amino-allyl modified dUTP in a reverse transcription reaction and coupled to reactive Cy3 or Cy5 dyes (Amersham). Correspondingly labeled Cy3 and Cy5 cDNA products were combined and purified on PCR purification columns (Qiagen), and diluted in hybridization buffer. Probes were added to the slides and incubated in a metal hybidization chamber (DieTech) in a waterbath at 65°C over night. Microarrays were washed and scanned on a GenePix 4000A scanner (Axon Instruments, Foster City, CA). Signal intensities were initially stored and analyzed using NOMAD (www.microarrays.org). Data was extracted as the global median normalized ratios (net median ratios) of the intensitites and log2 transformed. Signals with intensity values of the sum of median (foreground minus background) smaller than 300 were eliminated.  Each comparison was performed in replicates with independently isolated and processed probes. Within a set of replicate experiments, dyes were reversed to avoid bias in about half of the experiments. To represent all experiments with the same dye orientation the values of the log2 transformed expression ratios were reversed if necessary.

source_name: Wingless over-expressing prothoracic leg imaginal disc


title: Transdetermination study; D(wg) cells vs V(wg) cells_7-048

description: These experiments are described in Klebes et al. (Development, 2005, in press).  Comparisons – category #6,  slide 7-048,  V(wg) cells – probe 1 D(wg) cells – probe 2; (ratios are shown as probe1/probe2). Unique cell populations of Wingless over-expressing prothoracic leg imaginal discs of y,w,hs-flp; vgBE-GAL4/UASGFP, Actinc5, FRT y+, FRT, wg+ Drosophila larvae were compared with each other. In this experiment, dorsal cells (D(wg)) were compared to ventral cells (V(wg)) isolated from the same discs. These comparisons were intended to establish the unique expression profiles of dorsal and ventral leg imaginal cells in response to Wingless over-expression and serve as control experiments for the transdetermination study.  Prothoracic leg imaginal disc cells of the “weak point” that are competent to undergo transdetermination (TD cells) were labeled in vivo using the vgBE::GAL4/UAS-GFP reporter system (Sustar and Schubiger, 2005). Transdetermination was induced in these discs by over-expressing Wingless. TD cells were micro-dissected under a fluorescent dissecting scope. The remaining cells of the leg disc were separated into a dorsal (D(wg)) that lack the TD cells and ventral (V(wg)) fraction by cutting approximately along the median of the disc. Pooled fragments from 4-8 discs were used. Note that the D(wg) cell population represents all non-GFP-expressing dorsal cell of the leg disc. Ectopic Wg expression was induced by heat shock of young L3 larvae 60 h and 72h after egg deposition, and leg discs were isolated three days later. This protocol results in Wg over-expression in over 90% of the disc cells after heat shock (Maves and Schubiger, 1995). Note that Wg over-expression delays metamorphosis by one day, so these larvae had not yet pupariated. Discs and fragments were dissected in PBS and carefully washed several times to reduce the concentration of contaminating fat body cells.   RNA was extracted from imaginal disc fragments using the Mini RNA isolation kit (Zymo research). Linear RNA amplification was performed essentially as described (Klebes et al., 2002). In brief, total RNA was used for a first round of reverse transcription and in vitro transcription using T7 RNA polymerase. Subsequently, the amplified RNA product was subjected to a 2nd round of reverse transcription and in vitro transcription yielding 10,000-fold amplification or more.   Probes were indirectly labeled with amino-allyl modified dUTP in a reverse transcription reaction and coupled to reactive Cy3 or Cy5 dyes (Amersham). Correspondingly labeled Cy3 and Cy5 cDNA products were combined and purified on PCR purification columns (Qiagen), and diluted in hybridization buffer. Probes were added to the slides and incubated in a metal hybidization chamber (DieTech) in a waterbath at 65°C over night. Microarrays were washed and scanned on a GenePix 4000A scanner (Axon Instruments, Foster City, CA). Signal intensities were initially stored and analyzed using NOMAD (www.microarrays.org). Data was extracted as the global median normalized ratios (net median ratios) of the intensitites and log2 transformed. Signals with intensity values of the sum of median (foreground minus background) smaller than 300 were eliminated.  Each comparison was performed in replicates with independently isolated and processed probes. Within a set of replicate experiments, dyes were reversed to avoid bias in about half of the experiments. To represent all experiments with the same dye orientation the values of the log2 transformed expression ratios were reversed if necessary.


title: Transdetermination study; TD cells vs D(wg) cells_7-210

description: These experiments are described in Klebes et al. (Development, 2005, in press).  Comparisons – category #4,  slide 7-210, TD cells – probe 1 vs. D(wg) cells – probe 2, (ratios are shown as probe2/probe1).  Unique cell populations of Wingless over-expressing prothoracic leg imaginal discs of y,w,hs-flp; vgBE-GAL4/UASGFP, Actinc5, FRT y+, FRT, wg+ Drosophila larvae were compared with each other. In this experiment, cells that undergo transdetermination (TD cells) were compared to dorsal cells (D(wg)) isolated from the same discs. These comparisons were intended to establish the unique expression profiles of leg imaginal cells (TD cells) while they undergo a fate switch to transdetermine into wing cells.  Prothoracic leg imaginal disc cells of the “weak point” that are competent to undergo transdetermination (TD cells) were labeled in vivo using the vgBE::GAL4/UAS-GFP reporter system (Sustar and Schubiger, 2005). Transdetermination was induced in these discs by over-expressing Wingless. TD cells were micro-dissected under a fluorescent dissecting scope. The remaining cells of the leg disc were separated into a dorsal (D(wg)) that lack the TD cells and ventral (V(wg)) fraction by cutting approximately along the median of the disc. Pooled fragments from 4-8 discs were used. Note that the D(wg) cell population represents all non-GFP-expressing dorsal cell of the leg disc.  Ectopic Wg expression was induced by heat shock of young L3 larvae 60 h and 72h after egg deposition, and leg discs were isolated three days later. This protocol results in Wg over-expression in over 90% of the disc cells after heat shock (Maves and Schubiger, 1995). Note that Wg over-expression delays metamorphosis by one day, so these larvae had not yet pupariated. Discs and fragments were dissected in PBS and carefully washed several times to reduce the concentration of contaminating fat body cells.   RNA was extracted from imaginal disc fragments using the Mini RNA isolation kit (Zymo research). Linear RNA amplification was performed essentially as described (Klebes et al., 2002). In brief, total RNA was used for a first round of reverse transcription and in vitro transcription using T7 RNA polymerase. Subsequently, the amplified RNA product was subjected to a 2nd round of reverse transcription and in vitro transcription yielding 10,000-fold amplification or more.   Probes were indirectly labeled with amino-allyl modified UTP in a reverse transcription reaction and coupled to reactive Cy3 or Cy5 dyes (Amersham). Correspondingly labeled Cy3 and Cy5 cDNA products were combined and purified on PCR purification columns (Qiagen), and diluted in hybridization buffer. Probes were added to the slides and incubated in a metal hybidization chamber (DieTech) in a waterbath at 65°C over night. Microarrays were washed and scanned on a GenePix 4000A scanner (Axon Instruments, Foster City, CA). Signal intensities were initially stored and analyzed using NOMAD (www.microarrays.org). Data was extracted as the global median normalized ratios (net median ratios) of the intensitites and log2 transformed. Signals with intensity values of the sum of median (foreground minus background) smaller than 300 were eliminated.  Each comparison was performed in replicates with independently isolated and processed probes. Within a set of replicate experiments, dyes were reversed to avoid bias in about half of the experiments. To represent all experiments with the same dye orientation the values of the log2 transformed expression ratios were reversed if necessary.


title: Transdetermination study; wing vs leg_5-256

description: These experiments are described in Klebes et al. (Development, 2005, in press). Comparisons – category #2, slide 5-256, prothoracic leg disc – probe 1 vs. wing disc – probe 2. (ratios are shown as probe1/probe2). Wing imaginal discs of single wildtype Drosophila larvae were compared to a single leg imaginal disc of the same animal. In this experiment the leg disc was a prothoracic (1st) leg imaginal disc. These comparisons were intended to establish the unique expression profiles of wing and leg imaginal discs. For this experiment wing and leg imaginal discs of third instar larvae of Drosophila melanogaster strain OregonR (OR) were staged by feeding 1% bromphenol blue which allows to observe the gut content. Single male wandering late third instar larvae (half empty gut) were used to isolate wing and leg discs. Discs were dissected in PBS and carefully washed several times to reduce the concentration of contaminating fat body cells. RNA was extracted from single imaginal discs using the Mini RNA isolation kit (Zymo research). Linear RNA amplification was performed essentially as described (Klebes et al., 2002). In brief, total RNA was used for a first round of reverse transcription and in vitro transcription using T7 RNA polymerase. Subsequently, the amplified RNA product was subjected to a 2nd round of reverse transcription and in vitro transcription yielding 10,000-fold amplification or more. Probes were indirectly labeled with amino-allyl modified dUTP in a reverse transcription reaction and coupled to reactive Cy3 or Cy5 dyes (Amersham). Correspondingly labeled Cy3 and Cy5 cDNA products were combined and purified on PCR purification columns (Qiagen), and diluted in hybridization buffer. Probes were added to the slides and incubated in a metal hybidization chamber (DieTech) in a waterbath at 65°C over night. Microarrays were washed and scanned on a GenePix 4000A scanner (Axon Instruments, Foster City, CA). Signal intensities were initially stored and analyzed using NOMAD (www.microarrays.org). Data was extracted as the global median normalized ratios (net median ratios) of the intensitites and log2 transformed. Signals with intensity values of the sum of median (foreground minus background) smaller than 300 were eliminated. Each comparison was performed in replicates with independently isolated and processed probes. Within a set of replicate experiments, dyes were reversed to avoid bias in about half of the experiments. To represent all experiments with the same dye orientation the values of the log2 transformed expression ratios were reversed if necessary.

source_name: prothoracic leg imaginal disc

tag: wildtype (OR) prothoracic leg imaginal disc of third instar male larva


title: Transdetermination study; wing vs leg_5-249

description: These experiments are described in Klebes et al. (Development, 2005, in press). Comparisons – category #2, slide 5-249, mesothoracic leg disc – probe 1 vs. wing disc – probe 2. (ratios are shown as probe1/probe2). Wing imaginal discs of single wildtype Drosophila larvae were compared to a single leg imaginal disc of the same animal. In this experiment the leg disc was a mesothoracic (2nd) leg imaginal disc. These comparisons were intended to establish the unique expression profiles of wing and leg imaginal discs. For this experiment wing and leg imaginal discs of third instar larvae of Drosophila melanogaster strain OregonR (OR) were staged by feeding 1% bromphenol blue which allows to observe the gut content. Single male wandering late third instar larvae (half empty gut) were used to isolate wing and leg discs. Discs were dissected in PBS and carefully washed several times to reduce the concentration of contaminating fat body cells. RNA was extracted from single imaginal discs using the Mini RNA isolation kit (Zymo research). Linear RNA amplification was performed essentially as described (Klebes et al., 2002). In brief, total RNA was used for a first round of reverse transcription and in vitro transcription using T7 RNA polymerase. Subsequently, the amplified RNA product was subjected to a 2nd round of reverse transcription and in vitro transcription yielding 10,000-fold amplification or more. Probes were indirectly labeled with amino-allyl modified dUTP in a reverse transcription reaction and coupled to reactive Cy3 or Cy5 dyes (Amersham). Correspondingly labeled Cy3 and Cy5 cDNA products were combined and purified on PCR purification columns (Qiagen), and diluted in hybridization buffer. Probes were added to the slides and incubated in a metal hybidization chamber (DieTech) in a waterbath at 65°C over night. Microarrays were washed and scanned on a GenePix 4000A scanner (Axon Instruments, Foster City, CA). Signal intensities were initially stored and analyzed using NOMAD (www.microarrays.org). Data was extracted as the global median normalized ratios (net median ratios) of the intensitites and log2 transformed. Signals with intensity values of the sum of median (foreground minus background) smaller than 300 were eliminated. Each comparison was performed in replicates with independently isolated and processed probes. Within a set of replicate experiments, dyes were reversed to avoid bias in about half of the experiments. To represent all experiments with the same dye orientation the values of the log2 transformed expression ratios were reversed if necessary.

source_name: mesothoracic leg imaginal disc

tag: wildtype (OR) mesothoracic leg imaginal disc of third instar male larva


title: Transdetermination study; wing vs leg_6-59

description: These experiments are described in Klebes et al. (Development, 2005, in press).  Comparisons – category #2,  slide 6-59, wing disc – probe 1 vs. prothoracic leg disc – probe 2. (ratios are shown as probe2/probe1).  Wing imaginal discs of single wildtype Drosophila larvae were compared to a single leg imaginal disc of the same animal. In this experiment the leg disc was a prothoracic (1st) leg imaginal disc. These comparisons were intended to establish the unique expression profiles of wing and leg imaginal discs. For this experiment wing and leg imaginal discs of third instar larvae of Drosophila melanogaster strain OregonR (OR) were staged by feeding 1% bromphenol blue which allows to observe the gut content. Single male wandering late third instar larvae (half empty gut) were used to isolate wing and leg discs. Discs were dissected in PBS and carefully washed several times to reduce the concentration of contaminating fat body cells.   RNA was extracted from single imaginal discs using the Mini RNA isolation kit (Zymo research). Linear RNA amplification was performed essentially as described (Klebes et al., 2002). In brief, total RNA was used for a first round of reverse transcription and in vitro transcription using T7 RNA polymerase. Subsequently, the amplified RNA product was subjected to a 2nd round of reverse transcription and in vitro transcription yielding 10,000-fold amplification or more.   Probes were indirectly labeled with amino-allyl modified dUTP in a reverse transcription reaction and coupled to reactive Cy3 or Cy5 dyes (Amersham). Correspondingly labeled Cy3 and Cy5 cDNA products were combined and purified on PCR purification columns (Qiagen), and diluted in hybridization buffer. Probes were added to the slides and incubated in a metal hybidization chamber (DieTech) in a waterbath at 65°C over night. Microarrays were washed and scanned on a GenePix 4000A scanner (Axon Instruments, Foster City, CA). Signal intensities were initially stored and analyzed using NOMAD (www.microarrays.org). Data was extracted as the global median normalized ratios (net median ratios) of the intensitites and log2 transformed. Signals with intensity values of the sum of median (foreground minus background) smaller than 300 were eliminated.  Each comparison was performed in replicates with independently isolated and processed probes. Within a set of replicate experiments, dyes were reversed to avoid bias in about half of the experiments. To represent all experiments with the same dye orientation the values of the log2 transformed expression ratios were reversed if necessary.

treatment_protocol: dissected disc, 2 rounds of linear amplification


title: Transdetermination study; wing vs leg_6-62

description: These experiments are described in Klebes et al. (Development, 2005, in press).  Comparisons – category #2,  slide 6-62, mesothoracic leg disc – probe 1 vs. wing disc – probe 2. (ratios are shown as probe1/probe2).  Wing imaginal discs of single wildtype Drosophila larvae were compared to a single leg imaginal disc of the same animal. In this experiment the leg disc was a mesothoracic (2nd) leg imaginal disc. These comparisons were intended to establish the unique expression profiles of wing and leg imaginal discs. For this experiment wing and leg imaginal discs of third instar larvae of Drosophila melanogaster strain OregonR (OR) were staged by feeding 1% bromphenol blue which allows to observe the gut content. Single male wandering late third instar larvae (half empty gut) were used to isolate wing and leg discs. Discs were dissected in PBS and carefully washed several times to reduce the concentration of contaminating fat body cells.   RNA was extracted from single imaginal discs using the Mini RNA isolation kit (Zymo research). Linear RNA amplification was performed essentially as described (Klebes et al., 2002). In brief, total RNA was used for a first round of reverse transcription and in vitro transcription using T7 RNA polymerase. Subsequently, the amplified RNA product was subjected to a 2nd round of reverse transcription and in vitro transcription yielding 10,000-fold amplification or more.   Probes were indirectly labeled with amino-allyl modified dUTP in a reverse transcription reaction and coupled to reactive Cy3 or Cy5 dyes (Amersham). Correspondingly labeled Cy3 and Cy5 cDNA products were combined and purified on PCR purification columns (Qiagen), and diluted in hybridization buffer. Probes were added to the slides and incubated in a metal hybidization chamber (DieTech) in a waterbath at 65°C over night. Microarrays were washed and scanned on a GenePix 4000A scanner (Axon Instruments, Foster City, CA). Signal intensities were initially stored and analyzed using NOMAD (www.microarrays.org). Data was extracted as the global median normalized ratios (net median ratios) of the intensitites and log2 transformed. Signals with intensity values of the sum of median (foreground minus background) smaller than 300 were eliminated.  Each comparison was performed in replicates with independently isolated and processed probes. Within a set of replicate experiments, dyes were reversed to avoid bias in about half of the experiments. To represent all experiments with the same dye orientation the values of the log2 transformed expression ratios were reversed if necessary.


title: dam mutS mutant replicate 2

status: Public on Jul 14 2005

city: Cambridge

first: Jennifer

last: Robbins

state: MA

address: 77 Massachusetts Ave.

department: Biological Engineering

country: USA

institute: Massachusetts Institute of Technology

description: Overnight culture was diluted 1000-fold with fresh LB medium and cultured further.  Log phase culture was diluted to a cell density of 2 X 108 cells/ml in M9 salts and incubated at 37°C for two hours, after which it was resuspended in LB broth for 90 minutes.  This treatment served as a mock treatment for cultures incubated with a chemical in a parallel study. Total RNA was isolated from cells by extraction using the MasterPure RNA Purification Kit (Epicentre Technologies) followed by DNAse digestion according to the manufacturer’s protocol.  The isolated total RNA was quantitated by absorption at 260 nm (typical yield from a 15 ml culture was 250-500 µg of total RNA), and the purity was determined by the ratio of absorption values at 260/280nm.  RNA quality was determined by formaldehyde agarose gel electrophoresis (1.2% agarose in FA Buffer pH 7.0 (20 mM 3-[N-morpholino]propanesulfonic acid, 5 mM sodium acetate, 1 mM ethylenadiaminetetraacetic acide (EDTA))) or by analysis on an Agilent 2100 Bioanalyzer.  All samples visualized by gel electrophoresis or by the bioanalyzer electropherogram showed clear distinct bands correlating to 16S and 23S ribosomal RNA, indicating that no detectable RNA degradation occurred and that RNA integrity was maintained throughout the RNA isolation procedure. mRNA was enriched from total RNA as described in the Affymetrix GeneChip Expression Analysis Technical Manual for GeneChip E. coli Sense Genome Arrays.

hyb_protocol: Affymetrix

scan_protocol: Affymetrix

data_processing: RMA Version 0.1 Release

label: PEO-iodeacetyl-Biotin

organism: Escherichia coli

source_name: E. coli dam mutS mutant Strain GM5556

label_protocol: Affymetrix E. coli Sense arrays

growth_protocol: Exponentially growing culture held in M9 minimal salts for 120 min. then allowed to recover in LB for 90 min., after which total RNA was isolated.

extract_protocol: Epicentre MasterPure RNA Purification kit and Affymetrix protocol for E. coli Sense arrays (mRNA enrichment protocol)

tag: As GM3819 dam-16::Kan   but mutS215::Tn10


title: Transdetermination study; wing vs leg_5-248

description: These experiments are described in Klebes et al. (Development, 2005, in press).  Comparisons – category #2,  slide 5-248, wing disc – probe 1 vs. metathoracic leg disc – probe 2. (ratios are shown as probe2/probe1).  Wing imaginal discs of single wildtype Drosophila larvae were compared to a single leg imaginal disc of the same animal. In this experiment the leg disc was a metathoracic (3rd) leg imaginal disc. These comparisons were intended to establish the unique expression profiles of wing and leg imaginal discs. For this experiment wing and leg imaginal discs of third instar larvae of Drosophila melanogaster strain OregonR (OR) were staged by feeding 1% bromphenol blue which allows to observe the gut content. Single male wandering late third instar larvae (half empty gut) were used to isolate wing and leg discs. Discs were dissected in PBS and carefully washed several times to reduce the concentration of contaminating fat body cells.   RNA was extracted from single imaginal discs using the Mini RNA isolation kit (Zymo research). Linear RNA amplification was performed essentially as described (Klebes et al., 2002). In brief, total RNA was used for a first round of reverse transcription and in vitro transcription using T7 RNA polymerase. Subsequently, the amplified RNA product was subjected to a 2nd round of reverse transcription and in vitro transcription yielding 10,000-fold amplification or more.   Probes were indirectly labeled with amino-allyl modified dUTP in a reverse transcription reaction and coupled to reactive Cy3 or Cy5 dyes (Amersham). Correspondingly labeled Cy3 and Cy5 cDNA products were combined and purified on PCR purification columns (Qiagen), and diluted in hybridization buffer. Probes were added to the slides and incubated in a metal hybidization chamber (DieTech) in a waterbath at 65°C over night. Microarrays were washed and scanned on a GenePix 4000A scanner (Axon Instruments, Foster City, CA). Signal intensities were initially stored and analyzed using NOMAD (www.microarrays.org). Data was extracted as the global median normalized ratios (net median ratios) of the intensitites and log2 transformed. Signals with intensity values of the sum of median (foreground minus background) smaller than 300 were eliminated.  Each comparison was performed in replicates with independently isolated and processed probes. Within a set of replicate experiments, dyes were reversed to avoid bias in about half of the experiments. To represent all experiments with the same dye orientation the values of the log2 transformed expression ratios were reversed if necessary.


title: Transdetermination study; wing vs leg_6-64

description: These experiments are described in Klebes et al. (Development, 2005, in press).  Comparisons – category #2,  slide 6-64, wing disc – probe 1 vs. metathoracic leg disc – probe 2. (ratios are shown as probe2/probe1).  Wing imaginal discs of single wildtype Drosophila larvae were compared to a single leg imaginal disc of the same animal. In this experiment the leg disc was a metathoracic (3rd) leg imaginal disc. These comparisons were intended to establish the unique expression profiles of wing and leg imaginal discs. For this experiment wing and leg imaginal discs of third instar larvae of Drosophila melanogaster strain OregonR (OR) were staged by feeding 1% bromphenol blue which allows to observe the gut content. Single male wandering late third instar larvae (half empty gut) were used to isolate wing and leg discs. Discs were dissected in PBS and carefully washed several times to reduce the concentration of contaminating fat body cells.   RNA was extracted from single imaginal discs using the Mini RNA isolation kit (Zymo research). Linear RNA amplification was performed essentially as described (Klebes et al., 2002). In brief, total RNA was used for a first round of reverse transcription and in vitro transcription using T7 RNA polymerase. Subsequently, the amplified RNA product was subjected to a 2nd round of reverse transcription and in vitro transcription yielding 10,000-fold amplification or more.   Probes were indirectly labeled with amino-allyl modified dUTP in a reverse transcription reaction and coupled to reactive Cy3 or Cy5 dyes (Amersham). Correspondingly labeled Cy3 and Cy5 cDNA products were combined and purified on PCR purification columns (Qiagen), and diluted in hybridization buffer. Probes were added to the slides and incubated in a metal hybidization chamber (DieTech) in a waterbath at 65°C over night. Microarrays were washed and scanned on a GenePix 4000A scanner (Axon Instruments, Foster City, CA). Signal intensities were initially stored and analyzed using NOMAD (www.microarrays.org). Data was extracted as the global median normalized ratios (net median ratios) of the intensitites and log2 transformed. Signals with intensity values of the sum of median (foreground minus background) smaller than 300 were eliminated.  Each comparison was performed in replicates with independently isolated and processed probes. Within a set of replicate experiments, dyes were reversed to avoid bias in about half of the experiments. To represent all experiments with the same dye orientation the values of the log2 transformed expression ratios were reversed if necessary.

tag: wildtype (OR) metahoracic leg imaginal disc of third instar male larva