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Sample GSM402494 Query DataSets for GSM402494
Status Public on Jan 01, 2010
Title Tadpole pancreas Control replication 4
Sample type RNA
 
Channel 1
Source name Tadpole, pancreas, Control
Organism Xenopus laevis
Characteristics development stage: tadpole [Nieuwkoop and Faber stage (NF) 55]
individual identifier: Control A (C1) 251757610005A1
tissue: pancreas
agent: untreated
Biomaterial provider Donald Brown,Carnegie Institution,3520 San Martin Dr,Baltimore,MD,21218 E-mail: brown@ciwemb.edu
Treatment protocol Xenopus tadpoles (NF55) were treated with 1 mM methimazole in 0.1 X MMR solution for 1 week to block the endogenous TH production and reduce the TH present in the system of the tadpole. They were then treated with 100 nM T3 in 1 mM methimazole and 0.1 x MMR for another 48h. Samples were collected at 12h, 24h and 48h of exposure. Tadpoles exposed to 1mM MMI and not to T3 were control animals and they were collected at the time of last sampling of T3 exposure. Tadpoles were euthanized with ice-cold MS222, excess water was blotted from the animals with tissue paper, small incision was made on the belly for quick fixation and samples were fixed in RNA-Later overnight at 4C and used for RNA extraction in TRIZOL reagent. Pancreas from 5-10 tadpoles were pooled together as one replicate and there were three replicates at each time of exposure.
Extracted molecule total RNA
Extraction protocol TRIZOL standard extraction method was used . The full protocol can be found in the Life Technologies CAT 15596. Homogenize tissue samples in 1 mL of TRIZOL reagent per 50-100 mg of tissue using 1.5 Pellet Pestile (Nalge Nunc International). The sample volume should not exceed 10% of the volume of TRIZOL Reagent used for homogenization. Incubate the homogenized samples for 5 minutes at room temperature to permit the complete dissociation of nucleoprotein complexes. Add 0.2 mL of chloroform per 1 mL of TRIZOL Reagent. Cap sample tubes securely. Shake tubes vigorously by hand for 15 seconds and incubate them at room temperature for 2 to 3 minutes. Centrifuge the samples at no more than 12,000 x g for 15 minutes at 4 degree. Following centrifugation, the mixture separates into a lower red, phenol-chloroform phase, an interphase, and a colorless upper aqueous phase. RNA remains exclusively in the aqueous phase. The volume of the aqueous phase is about 60% of the volume of TRIZOL Reagent used for homogenization. Transfer the aqueous phase to a fresh tube, and precipitate the RNA from the aqueous phase by mixing with isopropyl alcohol. Use 0.5 mL of isopropyl alcohol per 1 mL of TRIZOL Reagent used for the initial homogenization. Incubate samples at room temperature for 10 minutes and centrifuge at no more than 12,000 x g for 10 minutes at 4 degree. The RNA precipitate, often invisible before centrifugation, forms a gel-like pellet on the side and bottom of the tube. Remove the supernate. Wash the RNA pellet once with 75% ethanol, adding at least 1 mL of 75% ethanol per 1 mL of TRIZOL Reagent used for the initial homogenization. Mix the sample by vortexing and centrifuge at no more than 7,500 x g for 5 minutes at 4 degree. Redissolving the RNA at the end of procedure, briefly drying the RNA pellet.
Label Cy3
Label protocol Total RNA was labeled using Agilents Fluorescent Linear Amplification kit, according to manufacturers instructions (Product Number G2554A or P/N G2556-66002, Version 3.0, June 2002).
 
Channel 2
Source name Universal RNA
Organism Xenopus laevis
Characteristics reference: Universal Xenopus Laevis Reference RNA
Extracted molecule total RNA
Extraction protocol mRNAs were extracted with QuickPrep Micro mRNA Purification Kit from Amersham
Label Cy5
Label protocol Total RNA was labeled using Agilents Fluorescent Linear Amplification kit, according to manufacturers instructions (Product Number G2554A or P/N G2556-66002, Version 3.0, June 2002).
 
 
Hybridization protocol Agilent 60-mer oligo microarray processing protocol (SSC Wash/SureHyb Chamber set-up) V4.1, April 2004. Agilent Publication Number: G4140-90030 V4.1 APRIL 2004.
Scan protocol Slides were scanned with an Agilent DNA Microarray Scanner (model G2505-64120) at 100% and 10% PMT in both channels, with a scan resolution of 5um.
Description stage 55 control samples, No T3 Exposure
Data processing Data are extracted with Agilent Feature Extraction Software.The data were further processed with NIA ANOVA tool utilities.See http://lgsun.grc.nia.nih.gov/ANOVA for details.
The data is normalized with the following method:
(1) Take values from the columns gDyeNormSignal,rDyeNormSignal in the raw files and log-transform them using: log10(max(x,1)). These values will be referenced below as Xgi and Xri where i is array number.
(2) Take average of Xri's for the oligo among 16 arrays in the series: AverXr = average(Xri).
(3) For each array estimate Yi = Xgi-Xri+AverXr
(4) Round Yi to 4 decimal digits. This is used as the normalized VALUE.
More detailed information with error correction can be found at http://lgsun.grc.nia.nih.gov/ANOVA/help.html#arrayjoin
 
Submission date May 12, 2009
Last update date May 14, 2009
Contact name Donald Brown
E-mail(s) brown@ciwemb.edu
Organization name Carnegie Institution
Department Department of Embryology
Street address 3520 San Martin Dr
City Baltimore
State/province MD
ZIP/Postal code 21218
Country USA
 
Platform ID GPL8463
Series (2)
GSE16074 Thyroid Hormone (TH) Controls The Remodeling Of The Pancreas And The Liver, Part A
GSE16075 Thyroid Hormone (TH) Controls The Remodeling Of The Pancreas And The Liver

Data table header descriptions
ID_REF Feature Number (FeatureNum).
VALUE The normalized value described in the Data Processing section.

Data table
ID_REF VALUE
14 2.4307
15 1.9508
16 2.3574
17 3.2385
18 3.6970
19 4.0641
21 2.3162
22 2.8672
23 4.1187
25 1.9366
26 3.1797
27 3.1227
28 1.5349
29 1.7483
30 2.4863
32 3.7466
33 1.6922
36 2.1082
37 3.6599
38 3.4680

Total number of rows: 33206

Table truncated, full table size 413 Kbytes.




Supplementary file Size Download File type/resource
GSM402494.txt.gz 9.8 Mb (ftp)(http) TXT
Processed data included within Sample table

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