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Sample GSM1195185 Query DataSets for GSM1195185
Status Public on Jun 01, 2014
Title Myd88mutant_PVP_4d_3
Sample type RNA
 
Channel 1
Source name D.rerio_myd88-experiment_CR
Organism Danio rerio
Characteristics sample type: The CR (common reference) was a mixture of all RNA samples from this microarray study.
tissue: embryo
Treatment protocol Zebrafish embryos were manually dechorionated at 24 hpf and at 28 hpf they were micro-injected into the caudal vein with 200 CFU of M. marinum Mma20 bacteria suspended in PBS/2%PVP or mock-injected with PBS/2%PVP as a control. At 5 days post fertilization (4 d post infection) three single embryos per treatment group were snap-frozen in liquid nitrogen, and total RNA was isolated using TRIZOL reagent.
Growth protocol Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols (http://ZFIN.org). Embryos were grown at 28,5°C in egg water (60µg/ml Instant Ocean sea salts).
Extracted molecule total RNA
Extraction protocol Single embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at -80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent (Invitrogen) and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI (Roche Applied Science) to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit (Qiagen) according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab-on-chip analysis using the 2100 Bioanalyzer (Agilent Technologies).
Label Cy3
Label protocol Amino Allyl modified aRNA was synthesized in one amplification round from 1 ug of total RNA using the Amino Allyl MessageAmp™ II aRNA Amplification Kit (Ambion). Subsequently, 6 ug of Amino Allyl modified aRNA was used for coupling of monoreactive Cy3 or Cy5 dye (GE Healthcare) and column purified.
 
Channel 2
Source name Myd88mutant_PVP_4d_3
Organism Danio rerio
Characteristics sample type: MyD88 mutant; mock-injected with buffer into the caudal vein at 28 hpf; RNA isolated at 4d post injection
tissue: embryo
treatment: mock-injected with buffer into the caudal vein at 28 hpf
genotype: MyD88 mutant
Treatment protocol Zebrafish embryos were manually dechorionated at 24 hpf and at 28 hpf they were micro-injected into the caudal vein with 200 CFU of M. marinum Mma20 bacteria suspended in PBS/2%PVP or mock-injected with PBS/2%PVP as a control. At 5 days post fertilization (4 d post infection) three single embryos per treatment group were snap-frozen in liquid nitrogen, and total RNA was isolated using TRIZOL reagent.
Growth protocol Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols (http://ZFIN.org). Embryos were grown at 28,5°C in egg water (60µg/ml Instant Ocean sea salts).
Extracted molecule total RNA
Extraction protocol Single embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at -80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent (Invitrogen) and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI (Roche Applied Science) to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit (Qiagen) according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab-on-chip analysis using the 2100 Bioanalyzer (Agilent Technologies).
Label Cy5
Label protocol Amino Allyl modified aRNA was synthesized in one amplification round from 1 ug of total RNA using the Amino Allyl MessageAmp™ II aRNA Amplification Kit (Ambion). Subsequently, 6 ug of Amino Allyl modified aRNA was used for coupling of monoreactive Cy3 or Cy5 dye (GE Healthcare) and column purified.
 
 
Hybridization protocol The dual colour hybridization of the microarray chips was performed at the Microarray Department (MAD) of the University of Amsterdam (Amsterdam, The Netherlands) using the standard Agilent protocol.
Scan protocol The arrays were scanned with DNA Microarray Scanner G2565CA from Agilent Technologies. The arrays were scanned twice with 10% PMT and 100% PMT laser power.
Description Biological replicate 3 of 3: mutant embryos PVP control 4d
Data processing Microarray data was processed from raw data image files with Feature Extraction Software 9.5.3.1 (Agilent Technologies). The XDR function was used to extend the dynamic range. Processed data were subsequently imported into Rosetta Resolver 7.0 (Rosetta Biosoftware, Seattle, Washington) and subjected to default ratio error modelling. The Agilent Feature Extraction files contain the final log ratio data. The algorithms that were used to normalize the feature signal and calculate the log ratios are described in the Reference Guide for Agilent Feature Extraction Software v9.5. The standard settings as described for the GE2-v5_95 protocol in the Reference Guide were used. In order to calculate the normalized signal, the Rank Consistency Probes method was used and the Linear&LOWESSDyeNormFactor (= DyeNormalSignal/(BGSubSignal x LinearDyeNormFactor) was calculated as described on page 231 of the Reference Guide. The dye normalized signal was calculated as follows (page 232): DyeNormSignal = BGSubSignal x DyeNormFactor. The log10 ratio was subsequently calculated as follows (page 232): LogRatio = Log(rProcessedSignal/gProcessedSignal), where rProcessedSignal and gProcessedSignal are signals post dye normalization and post surrogate processing in the red and green channels, respectively. The surrogate values are calculated and used as the lowest limit of detection to replace the dye normalized signal when either the mean feature signal is less than the background signal or not significant when compared to the background signal, or when the mean signal is less than its background standard deviation (page 226).
 
Submission date Jul 24, 2013
Last update date Jun 01, 2014
Contact name Annemarie H. Meijer
E-mail(s) a.h.meijer@biology.leidenuniv.nl
Organization name Institute of Biology, Leiden University
Department Animal Sciences and Health
Street address Einsteinweg 55
City Leiden
ZIP/Postal code 2333 CC
Country Netherlands
 
Platform ID GPL15180
Series (2)
GSE49187 Autophagy regulator DRAM1 functions downstream of MYD88 in defense against tuberculosis (array)
GSE49188 Autophagy regulator DRAM1 functions downstream of MYD88 in defense against tuberculosis

Data table header descriptions
ID_REF
VALUE normalized log10 ratio Cy5/Cy3

Data table
ID_REF VALUE
1 -3.613801470e-001
2 0.000000000e+000
3 0.000000000e+000
4 -3.936069600e-001
5 0.000000000e+000
6 0.000000000e+000
7 0.000000000e+000
8 0.000000000e+000
9 0.000000000e+000
10 0.000000000e+000
11 0.000000000e+000
12 0.000000000e+000
13 0.000000000e+000
14 0.000000000e+000
15 0.000000000e+000
16 0.000000000e+000
17 0.000000000e+000
18 0.000000000e+000
19 0.000000000e+000
20 0.000000000e+000

Total number of rows: 180822

Table truncated, full table size 4178 Kbytes.




Supplementary file Size Download File type/resource
GSM1195185_252823310086_3u20b_S01_GE2_107_Sep09_1_2.txt.gz 48.4 Mb (ftp)(http) TXT
Processed data included within Sample table
Processed data provided as supplementary file

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