Postia placenta MAD-698 gene expression in different media
Ontology highlight
ABSTRACT:
ORGANISM(S): Postia placenta
SUBMITTER: Dan Cullen
PROVIDER: E-GEOD-12540 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Publications
Genome, transcriptome, and secretome analysis of wood decay fungus Postia placenta supports unique mechanisms of lignocellulose conversion.
Martinez Diego D Challacombe Jean J Morgenstern Ingo I Hibbett David D Schmoll Monika M Kubicek Christian P CP Ferreira Patricia P Ruiz-Duenas Francisco J FJ Martinez Angel T AT Kersten Phil P Hammel Kenneth E KE Vanden Wymelenberg Amber A Gaskell Jill J Lindquist Erika E Sabat Grzegorz G Bondurant Sandra Splinter SS Larrondo Luis F LF Canessa Paulo P Vicuna Rafael R Yadav Jagjit J Doddapaneni Harshavardhan H Subramanian Venkataramanan V Pisabarro Antonio G AG Lavín José L JL Oguiza José A JA Master Emma E Henrissat Bernard B Coutinho Pedro M PM Harris Paul P Magnuson Jon Karl JK Baker Scott E SE Bruno Kenneth K Kenealy William W Hoegger Patrik J PJ Kües Ursula U Ramaiya Preethi P Lucas Susan S Salamov Asaf A Shapiro Harris H Tu Hank H Chee Christine L CL Misra Monica M Xie Gary G Teter Sarah S Yaver Debbie D James Tim T Mokrejs Martin M Pospisek Martin M Grigoriev Igor V IV Brettin Thomas T Rokhsar Dan D Berka Randy R Cullen Dan D
Proceedings of the National Academy of Sciences of the United States of America 20090204 6
Brown-rot fungi such as Postia placenta are common inhabitants of forest ecosystems and are also largely responsible for the destructive decay of wooden structures. Rapid depolymerization of cellulose is a distinguishing feature of brown-rot, but the biochemical mechanisms and underlying genetics are poorly understood. Systematic examination of the P. placenta genome, transcriptome, and secretome revealed unique extracellular enzyme systems, including an unusual repertoire of extracellular glyco ...[more]
Similar Datasets
Project description:Transcript profiles of Postia placenta grown on media containing ball-milled aspen or ball-milled pine as the sole carbon source were analyzed. Array design was based on the DoE's Joint Genome Institute's gene models for P. placenta version 1. The research goal is to identtify genes essential for cellulose depolymerization. From a data set of 12,438 unique alleles, each NimbleGen (Madison, WI) array featured 10 unique 60mers per gene, all in triplicate. The dataset was manually annotated to include only the ‘best allelic model’ among CAZY-encoding genes. Total RNA was purified from medium containing ball-milled aspen or ball-milled pine as the sole carbon source. Three biological replicates per medium were used (6 separate arrays). RNA was converted to double-strand cDNA and labeled with the Cy3 fluorophore sample for hybridization to the Postia placenta MAD-698 whole genome expression array by Roche NimbleGen (Iceland). In brief, 10ug of total RNA was incubated with 1X first strand buffer, 10 mM DTT, 0.5mM dNTPs, 100 pM oligo T7 d(T)24 primer, and 200 units of SuperScript II (Invitrogen) for 60 min at 42°C. Second strand cDNA was synthesized by incubation with 1X second strand buffer, 0.2mM dNTPs, 0.07 units per ul DNA ligase (Invitrogen), 0.27 units per ul DNA polymerase I (Invitrogen), 0.013 units per ul RNase H (Invitrogen), at 16°C for 2 hours. Immediately following, 10 units T4 DNA polymerase (Invitrogen) was added for additional 5 minute incubation at 16°C. Double-stranded cDNA was treated with 27ng/ul of RNase A (EpiCenter Technologies) for 10 minutes at 37°C. Treated cDNA was purified using an equal volume of phenol:chloroform:isoamyl alcohol (Ambion), ethanol precipitated, washed with 80% ethanol, and resuspended in 20ul water. One ug of each cDNA sample was amplified and labeled with 1 unit per ul of Klenow Fragment (New England BioLabs) and 1 O.D unit of Cy3 fluorophore (TriLink Biotechnologies, Inc.) for 2 hours at 37°C. Array hybridization was carried out with 6ug of labeled cDNA suspended in NimbleGen hybridization solution for 17 hours at 42°C.
2011-06-02 | E-GEOD-29656 | biostudies-arrayexpress
Project description:Transcript profiles of Phanerochaete chrysosporium grown on carbon-limited medium, nitrogen-limited medium and replete medium. Array design based on the DoE's Joint Genome Institute's v2.1 annotation. Goal is to define genes involved in lignin degradation. Keywords: gene expression under three different culture conditions From a data set of 10,004 unique alleles, each Roche NimbleGen (Madison, WI) array featured 12 unique 60mer probes per gene, all in triplicate. (Seven gene models composed mostly of repetitive DNA were represented by only 2 to 11 60mers.) Total RNA was purified from the five abovementioned media. In short, cultures were harvested by filtering through Miracloth (Calbiochem, EMD Biosciences, Gibbstown, NJ), squeeze dried and snap frozen in liquid nitrogen. Pellets were stored at -80 C until use. Extraction buffer was prepared by combining 10 ml 690 mM para-aminosalicylic acid (sodium salt) (Sigma-Aldrich, St. Louis, MO) with 10 ml 56 mM triisopropylnapthalene sulfonic acid (sodium salt) (Sigma-Aldrich), and placed on ice. To this was added 5 ml 5X RNB (1.0 M Tris, 1.25 M NaCl, 0.25 M EGTA). The pH of the 5X RNB was adjusted to 8.5 with NaOH. The mixture was kept on ice and shaken just before use. Frozen fungal pellets were ground to a fine powder with liquid nitrogen in an acid washed, pre-chilled mortar and pestle. The ground mycelia were transferred to Falcon 2059 tubes (VWR International, West Chester, PA), and extraction buffer was added to make a thick slurry. The samples were vortexed vigorously and placed on ice until all samples were processed. One half volume TE-saturated phenol (Sigma-Aldrich) and ¼ volume chloroform (Sigma-Aldrich) were added to each sample and vortexed vigorously. Samples were spun at 2940 x g in a fixed-angle rotor for 5 minutes. The aqueous layer was removed to a new tube, and phenol:chloroform extractions were repeated until the interface between the aqueous and organic layers was clear. The final aqueous extractions were placed in clean 2059 tubes, to which was added 0.1 volume 3M sodium acetate, pH 5.2, (DEPC-treated) and 2 volumes absolute ethanol. The tubes were shaken vigorously and stored overnight at -20˚C. The tubes were spun 1 hour at 2940 x g, the supernatants were decanted, and the pellets were resuspended in 4 ml RNase-free H2O. Total RNA was purified using the RNeasy Maxi kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol for RNA cleanup. RNAs were eluted from the RNeasy spin columns using two spins, for a final volume of 2 ml. The eluted RNAs were ethanol precipitated and stored overnight at -20˚C. The RNAs were spun 1 hour at 2940 x g, washed 1x with 70% ethanol, and resuspended in 50-100ul RNase-free H2O. Three biological replicates per medium were used (15 separate arrays). RNA was converted to double-strand cDNA and labeled with the Cy3 fluorophore sample for hybridization to the array by Roche NimbleGen (Iceland). In brief, 10ug of total RNA was incubated with 1X first strand buffer, 10 mM DTT, 0.5mM dNTPs, 100 pM oligo T7 d(T)24 primer, and 400U of SuperScript II (Invitrogen) for 60 min at 42˚C. Second strand cDNA was synthesized by incubation with 1X second strand buffer, 0.2mM dNTPs, 0.07U/ul DNA ligase (Invitrogen), 0.27U/ul DNA polymerase I (Invitrogen), 0.013U/ul RNase H (Invitrogen), at 16˚C for 2 hours. Immediately following, 10U T4 DNA polymerase (Invitrogen) was added for additional 5 minute incubation at 16˚C. Double-stranded cDNA was treated with 27ng/ul of RNase A (EpiCenter Technologies) for 10 minutes at 37˚C. Treated cDNA was purified using an equal volume of phenol:chloroform:isoamyl alcohol (Ambion), ethanol precipitated, washed with 80% ethanol, and resuspended in 20ul water. One ug of each cDNA sample was amplified and labeled with 1 unit per ul of Klenow Fragment (New England BioLabs) and 1 O.D. unit of Cy3 fluorophore (TriLink Biotechnologies, Inc.) for 2 hours at 37˚C. Array hybridization was carried out with 6ug of labeled cDNA suspended in NimbleGen hybridization solution for 17 hours at 42˚C. Arrays were scanned on the Axon4000B Scanner (Molecular Dynamics) and data was extracted from the scanned image using NimbleScan v2.4. DNASTAR ArrayStar v2.1 (Madison,WI) software was used to quantify and visualize data. Analyses were based on three biological replicates per culture medium. Quantile normalization and robust multi-array averaging (RMA; ) were applied to the entire dataset.
2009-03-30 | E-GEOD-14735 | biostudies-arrayexpress
Project description:Transcript profiles of Phanerochaete chrysosporium grown on different substrates were analyzed. Array design based on the DoE's Joint Genome Institute's v2.1 annotation. Goal is to define genes involved in cellulose degradation. Keywords: gene expression under different culture conditions From a data set of 10,004 unique alleles, each Roche NimbleGen (Madison, WI) array featured 12 unique 60mer probes per gene, all in triplicate. (Seven gene models composed mostly of repetitive DNA were represented by only 2 to 11 60mers.) Total RNA was purified from below mentioned media. In short, cultures were harvested by filtering through Miracloth (Calbiochem, EMD Biosciences, Gibbstown, NJ), squeeze dried and snap frozen in liquid nitrogen. Pellets were stored at -80 C until use. Extraction buffer was prepared by combining 10 ml 690 mM para-aminosalicylic acid (sodium salt) (Sigma-Aldrich, St. Louis, MO) with 10 ml 56 mM triisopropylnapthalene sulfonic acid (sodium salt) (Sigma-Aldrich), and placed on ice. To this was added 5 ml 5X RNB (1.0 M Tris, 1.25 M NaCl, 0.25 M EGTA). The pH of the 5X RNB was adjusted to 8.5 with NaOH. The mixture was kept on ice and shaken just before use. Frozen fungal pellets were ground to a fine powder with liquid nitrogen in an acid washed, pre-chilled mortar and pestle. The ground mycelia were transferred to Falcon 2059 tubes (VWR International, West Chester, PA), and extraction buffer was added to make a thick slurry. The samples were vortexed vigorously and placed on ice until all samples were processed. One half volume TE-saturated phenol (Sigma-Aldrich) and ¼ volume chloroform (Sigma-Aldrich) were added to each sample and vortexed vigorously. Samples were spun at 2940 x g in a fixed-angle rotor for 5 minutes. The aqueous layer was removed to a new tube, and phenol:chloroform extractions were repeated until the interface between the aqueous and organic layers was clear. The final aqueous extractions were placed in clean 2059 tubes, to which was added 0.1 volume 3M sodium acetate, pH 5.2, (DEPC-treated) and 2 volumes absolute ethanol. The tubes were shaken vigorously and stored overnight at -20˚C. The tubes were spun 1 hour at 2940 x g, the supernatants were decanted, and the pellets were resuspended in 4 ml RNase-free H2O. total RNA was purified using the RNeasy Maxi kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol for RNA cleanup. RNAs were eluted from the RNeasy spin columns using two spins, for a final volume of 2 ml. The eluted RNAs were ethanol precipitated and stored overnight at -20˚C. The RNAs were spun 1 hour at 2940 x g, washed 1x with 70% ethanol, and resuspended in 50-100ul RNase-free H2O. Three biological replicates per medium were used (15 separate arrays). RNA was converted to double-strand cDNA and labeled with the Cy3 fluorophore sample for hybridization to the array by Roche NimbleGen (Iceland). In brief, 10ug of total RNA was incubated with 1X first strand buffer, 10 mM DTT, 0.5mM dNTPs, 100 pM oligo T7 d(T)24 primer, and 400U of SuperScript II (Invitrogen) for 60 min at 42˚C. Second strand cDNA was synthesized by incubation with 1X second strand buffer, 0.2mM dNTPs, 0.07U/ul DNA ligase (Invitrogen), 0.27U/ul DNA polymerase I (Invitrogen), 0.013U/ul RNase H (Invitrogen), at 16˚C for 2 hours. Immediately following, 10U T4 DNA polymerase (Invitrogen) was added for additional 5 minute incubation at 16˚C. Double-stranded cDNA was treated with 27ng/ul of RNase A (EpiCenter Technologies) for 10 minutes at 37˚C. Treated cDNA was purified using an equal volume of phenol:chloroform:isoamyl alcohol (Ambion), ethanol precipitated, washed with 80% ethanol, and resuspended in 20ul water. One ug of each cDNA sample was amplified and labeled with 1 unit per ul of Klenow Fragment (New England BioLabs) and 1 O.D. unit of Cy3 fluorophore (TriLink Biotechnologies, Inc.) for 2 hours at 37˚C. Array hybridization was carried out with 6ug of labeled cDNA suspended in NimbleGen hybridization solution for 17 hours at 42˚C. Arrays were scanned on the Axon4000B Scanner (Molecular Dynamics) and data was extracted from the scanned image using NimbleScan v2.4. DNASTAR ArrayStar v2.1 (Madison,WI) software was used to quantify and visualize data. Analyses were based on three biological replicates per culture medium. Quantile normalization and robust multi-array averaging (RMA) were applied to the entire dataset.
2009-03-30 | E-GEOD-14734 | biostudies-arrayexpress
Project description:Transcript profiles of Phanerochaete chrysosporium grown on ball-milled aspen or ball-milled pine were analyzed. Array design based on the DoE's Joint Genome Institute's v2.1 annotation. Goal is to define genes involved in lignocellulose degradation. From a data set of 10,004 unique alleles, each Roche NimbleGen (Madison, WI) array featured 12 unique 60mer probes per gene, all in triplicate. (Seven gene models composed mostly of repetitive DNA were represented by only 2 to 11 60mers.) Total RNA was purified from medium containing ball-milled aspen or ball-milled pine as the sole carbon source. In short, cultures were harvested by filtering through Miracloth (Calbiochem, EMD Biosciences, Gibbstown, NJ), squeeze dried and snap frozen in liquid nitrogen. Pellets were stored at -80 C until use. Extraction buffer was prepared by combining 10 ml 690 mM para-aminosalicylic acid (sodium salt) (Sigma-Aldrich, St. Louis, MO) with 10 ml 56 mM triisopropylnapthalene sulfonic acid (sodium salt) (Sigma-Aldrich), and placed on ice. To this was added 5 ml 5X RNB (1.0 M Tris, 1.25 M NaCl, 0.25 M EGTA). The pH of the 5X RNB was adjusted to 8.5 with NaOH. The mixture was kept on ice and shaken just before use. Frozen fungal pellets were ground to a fine powder with liquid nitrogen in an acid washed, pre-chilled mortar and pestle. The ground mycelia were transferred to Falcon 2059 tubes (VWR International, West Chester, PA), and extraction buffer was added to make a thick slurry. The samples were vortexed vigorously and placed on ice until all samples were processed. One half volume TE-saturated phenol (Sigma-Aldrich) and M-BM-< volume chloroform (Sigma-Aldrich) were added to each sample and vortexed vigorously. Samples were spun at 2940 x g in a fixed-angle rotor for 5 minutes. The aqueous layer was removed to a new tube, and phenol:chloroform extractions were repeated until the interface between the aqueous and organic layers was clear. The final aqueous extractions were placed in clean 2059 tubes, to which was added 0.1 volume 3M sodium acetate, pH 5.2, (DEPC-treated) and 2 volumes absolute ethanol. The tubes were shaken vigorously and stored overnight at -20M-KM-^ZC. The tubes were spun 1 hour at 2940 x g, the supernatants were decanted, and the pellets were resuspended in 4 ml RNase-free H2O. Total RNA was purified using the RNeasy Maxi kit (Qiagen, Valencia, CA) according to the manufacturerM-bM-^@M-^Ys protocol for RNA cleanup. RNAs were eluted from the RNeasy spin columns using two spins, for a final volume of 2 ml. The eluted RNAs were ethanol precipitated and stored overnight at -20M-KM-^ZC. The RNAs were spun 1 hour at 2940 x g, washed 1x with 70% ethanol, and resuspended in 50-100ul RNase-free H2O. Three biological replicates per medium were used (6 separate arrays). RNA was converted to double-strand cDNA and labeled with the Cy3 fluorophore sample for hybridization to the array by Roche NimbleGen (Iceland). In brief, 10ug of total RNA was incubated with 1X first strand buffer, 10 mM DTT, 0.5mM dNTPs, 100 pM oligo T7 d(T)24 primer, and 400U of SuperScript II (Invitrogen) for 60 min at 42M-KM-^ZC. Second strand cDNA was synthesized by incubation with 1X second strand buffer, 0.2mM dNTPs, 0.07U/ul DNA ligase (Invitrogen), 0.27U/ul DNA polymerase I (Invitrogen), 0.013U/ul RNase H (Invitrogen), at 16M-KM-^ZC for 2 hours. Immediately following, 10U T4 DNA polymerase (Invitrogen) was added for additional 5 minute incubation at 16M-KM-^ZC. Double-stranded cDNA was treated with 27ng/ul of RNase A (EpiCenter Technologies) for 10 minutes at 37M-KM-^ZC. Treated cDNA was purified using an equal volume of phenol:chloroform:isoamyl alcohol (Ambion), ethanol precipitated, washed with 80% ethanol, and resuspended in 20ul water. One ug of each cDNA sample was amplified and labeled with 1 unit per ul of Klenow Fragment (New England BioLabs) and 1 O.D. unit of Cy3 fluorophore (TriLink Biotechnologies, Inc.) for 2 hours at 37M-KM-^ZC. Array hybridization was carried out with 6ug of labeled cDNA suspended in NimbleGen hybridization solution for 17 hours at 42M-KM-^ZC. Arrays were scanned on the Axon4000B Scanner (Molecular Dynamics) and data was extracted from the scanned image using NimbleScan v2.4. DNASTAR ArrayStar v2.1 (Madison,WI) software was used to quantify and visualize data. Analyses were based on three biological replicates per culture medium. Quantile normalization and robust multi-array averaging (RMA) were applied to the entire dataset.
2011-06-02 | E-GEOD-27941 | biostudies-arrayexpress
Project description:Transscript profiles of Ceriporiopsis subvermispora grown on different substrates were analyszed. Array design was based on the DoE's Joint Genome Institute's gene models for C. subvermispora version 1. The research goal is to identtify genes essential for lignocellulose depolymerization. From a data set of 12,125 unique gene models, each NimbleGen (Madison, WI) array targeted 12083 genes and featured 8 unique 60mers per gene, all in quadruplicate. Total RNA was purified from 5-day old cultures containing microcrystalline cellulose (Avicel) or glucose or ball-milled Populus as sole carbon source. Three biological replicates per medium were used (9 separate arrays). In short, cultures were harvested by filtering through Miracloth (Calbiochem, EMD Biosciences, Gibbstown, NJ), squeeze dried and snap frozen in liquid nitrogen. Pellets were stored at -80 C until use. Extraction buffer was prepared by combining 10 ml 690 mM para-aminosalicylic acid (sodium salt) (Sigma-Aldrich, St. Louis, MO) with 10 ml 56 mM triisopropylnapthalene sulfonic acid (sodium salt) (Sigma-Aldrich), and placed on ice. To this was added 5 ml 5X RNB (1.0 M Tris, 1.25 M NaCl, 0.25 M EGTA). The pH of the 5X RNB was adjusted to 8.5 with NaOH. The mixture was kept on ice and shaken just before use. Frozen fungal pellets were ground to a fine powder with liquid nitrogen in an acid washed, pre-chilled mortar and pestle. The ground mycelia were transferred to Falcon 2059 tubes (VWR International, West Chester, PA), and extraction buffer was added to make a thick slurry. The samples were vortexed vigorously and placed on ice until all samples were processed. One half volume TE-saturated phenol (Sigma-Aldrich) and ¼ volume chloroform (Sigma-Aldrich) were added to each sample and vortexed vigorously. Samples were spun at 2940 x g in a fixed-angle rotor for five minutes. The aqueous layer was removed to a new tube, and phenol:chloroform extractions were repeated until the interface between the aqueous and organic layers was clear. The final aqueous extractions were placed in clean 2059 tubes, to which was added 0.1 volume 3M sodium acetate, pH 5.2, (DEPC-treated) and two volumes absolute ethanol. The tubes were shaken vigorously and stored overnight at -20 0C. The tubes were spun 1 hour at 2940 x g, the supernatants were decanted, and the pellets were resuspended in 4 ml RNase-free H2O. Total RNA was purified using the RNeasy Maxi kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol for RNA cleanup. RNAs were eluted from the RNeasy spin columns using two spins, for a final volume of 2 ml. The eluted RNAs were ethanol precipitated and stored overnight at -20C. The RNAs were spun one hour at 2940 x g, washed once with 70% ethanol, and resuspended in 50-100 ?l RNase-free H2O. Three biological replicates per medium were used (nine separate arrays). RNA was converted to double-strand cDNA using Invitrogen's ds cDNA Synthesis kit (Carlsbad, CA) and labeled with the Cy3 fluorophore sample for hybridization to the array by Roche NimbleGen (Iceland). In brief, 10 ?g of total RNA was incubated with 1X first strand buffer, 10 mM DTT, 0.5 mM dNTPs, 100pM oligo T7 d(T)24 primer, and 400U of SuperScript II (Invitrogen) for 60 min at 42°C. Second strand cDNA was synthesized by incubation with 1X second strand buffer, 0.2 mM dNTPs, 0.07 U/ul DNA ligase (Invitrogen, Carlsbad, CA), 0.27 U/ul DNA polymerase I (Invitrogen, Carlsbad, CA), 0.013 U/ul RNase H (Invitrogen), at 16°C for two hours. Immediately following, 10U T4 DNA polymerase (Invitrogen) was added for an additional five minute incubation at 16°C. Double-stranded cDNA was treated with 27 ng/ul of RNase A (EpiCentre Technologies, Madison, WI) for 10 min at 37°C. Treated cDNA was purified using an equal volume of phenol:chloroform:isoamyl alcohol (Ambion/Life Technologies, Grand Island, NY), ethanol precipitated, washed with 80% ethanol, and resuspended in 20 ?l water. One ?g of each cDNA sample was amplified and labeled with one unit per ?l of Klenow Fragment (New England BioLabs, UK) and one OD unit of Cy3 fluorophore (TriLink Biotechnologies, Inc., San Diego, CA) for two hours at 37°C. Array hybridization was carried out with 6 ?g of labeled cDNA suspended in NimbleGen hybridization solution for 17 hours at 42°C. Arrays were scanned on the Axon 4000B Scanner (Molecular Devices, Sunnyvale, CA) and data was extracted from the scanned image using NimbleScan v2.4. DNASTAR ArrayStar v4 (Madison, WI) software was used to quantify and visualize data. .
2012-03-05 | E-GEOD-34636 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the following subset Series: GSE27941: Lignocellulose-induced regulation of Phanaerochaete chrysosporium genes GSE29656: Postia placenta MAD-698 gene expression in ball-milled aspen or ball-milled pine medium Refer to individual Series
2011-06-02 | E-GEOD-29659 | biostudies-arrayexpress
Project description:Two Chinese cabbage (Brassica rapa ssp. pekinensis) inbred lines, Chiifu (smaple A) and Kenshin (Smaple B), were grown for approximately 4 weeks in a growth chamber at 22degreesC under a 16 h light/8 h dark photoperiod with a photon flux density of 140 umol m-2 s-1. For microarray analysis, plants were exposed to a temperature of -4degreesC for 4 h, and shoots were sampled. Total RNA was isolated from the samples using an Easy-BLUETM Total RNA Extraction Kit (Invitrogen, NY, U.S.A.) and was then purified using an RNeasy MinEluteTM Cleanup Kit (Qiagen, Germany). Mircroarray experiments were carried out using Brassica 24K Oligo Microarray. RNA were prepared from each plant sample and 10 ug of total RNA were used for cDNA synthesis by using Superscript Double-Stranded cDNA Synthesis Kit (Invitrogen, NY, U.S.A.). Following cDNA synthesis, remaining RNA was removed by the addition of RNaseA and the cDNA was precipitated following phenol/chloroform extraction. The cDNA pellet was rehydrated and used for Cy3-labelling. For the synthesis of Cy3-labeled target DNA fragments, 1 ug of double strand cDNA was mixed with 40 ul (1 OD) of Cy3-9 mer primers (Sigma-Aldrich) and the total volume was adjusted to 80 ul with deionized water. After heating at 98degreesC for 10 min, 10 uL of dNTP mix (10 mM each) and 2 ul of Klenow fragment (NEB; 50 units/ul) were added and the reaction was incubated at 37degreesC for 2 h. Finally, the reaction was stopped by the addition of 10 ul of 0.5 M EDTA. Labeled DNA fragments were precipitated with isopropanol and rehydrated with water. The concentration of each sample was determined using spectrophotometer. For hybridization, 13 ug of the labeled DNA fragment was mixed with hybridization buffer (NimbleGen) and then hybridized with the microarray using a MAUI hybridization chamber (Biomicro) at 42degreesC for 16 h. Following hybridization, the microarray was washed with wash solution I, II, and III (NimbleGen), and then dried in a dark desiccator. The microarray slide was scanned using GenePix scanner 4000B (Axon) and the spot intensities were analyzed using a NimbleScan (NimbleGen). The normal distribution of Cy3 intensities was tested by qqline. The data was normalized and processed with cubic spline normalization using quantiles to adjust signal variations between chips and with Rubust Multi-Chip Analysis (RMA) using a median polish algorithm implemented in NimbleScan software (NimbleGen). Two biological replicates were carried out.
2014-04-24 | E-MTAB-1997 | biostudies-arrayexpress
Project description:We investigated the effects of PACAP knock out on the transcriptosome profile of adrenal gland. A 35,546-clone mouse cDNA microarray was used to investigate transcriptional changes Keywords: transcript identification design In the hybridization method employed, equal amounts (2-5 ug/15.5 ul) of total RNA obtained from adrenal gland of wild type and PACAP KO mice groups were separately labeled with two different fluorescent dyes (Cy3 or Cy5) and applied on the same NIMH Mouse 36K cDNA microarray chip containing 13217 distinct Entrez gene IDs (formerly known as LocusLink) and 15888 distinct unigene IDs, the remainders are incompletely annotated IMAGE clones. Probe preparation and hybridization were performed as follows. The total RNA (15-50 ug in 15.5 ul) was incubated with amine-modified random primer (2 ug/ul, 2 ul) and RNase inhibitor (5 units/ul, 1 ul) at 70C for 10 min. Primer-RNA solution was then incubated at 42C for 2 hr with the reverse transcriptase mix containing 5X first-strand buffer, 50X aa-dUTP/dNTPs (25 mM dATP, 25 mM dGTP, 25 mM dCTP, 15 mM dTTP and 10 mM aminoallyl dUTP), 0.1 M DDT, and Superscript II reverse transcriptase. The cDNA were labeled with NHS-ester Cy3 or Cy5 dye in the presence of 1 M sodium bicarbonate. Array slides placed in a hybridization chamber (Corning, Corning, NY) were incubated at 42C for 16-24 hr, and successively washed with 0.5X SSC, 0.01% SDS, and 0.06X SSC at room temperature for 10 min each. Arrays were scanned with a GenePix 4000A scanner (Axon, Foster City, CA), and the resulting images were analyzed using IPlab (Fairfax, VA) and a FileMaker Pro 5 (Santa Clara, CA) relational database designed by Zedong Chen, NHGRI. Following background subtraction and normalization, a calculated ratio of Cy3 to Cy5 signal intensities was used to define the relative increase or decrease of a particular transcript. Ratios were calculated only from those spots with a combined ratio quality above 0.3.
2010-05-26 | E-GEOD-7405 | biostudies-arrayexpress
Project description:Mass spectrometry raw data for secreted wood-degrading enzymes. Sample IDs are named as follows. ppl: Postia placenta, treesi: Trichoderma reesei, tvers: Trametes versicolor. Control: 1, oxidative treatment: 2. Biological replicates: a, b, c. For example, ppl_1a is Postia placenta, control sample, biological replicate a.
2020-06-26 | MSV000085635 | MassIVE
Project description:Transscript profiles of Postia placenta grown on different substrates were analyszed. Array design was based on the DoE's Joint Genome Institute's gene models for P. placenta version 1. The research goal is to identtify genes essential for cellulose depolymerization. Keywords: Culture condition comparison
2009-01-09 | GSE12540 | GEO