|
| Status |
Public on Jan 01, 2010 |
| Title |
Tadpole pancreas 24h replication 2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Tadpole, pancreas, TH, 24h, NF55
|
| Organism |
Xenopus laevis |
| Characteristics |
development stage: tadpole [Nieuwkoop and Faber stage (NF) 55]
individual identifier: 24H-B 251757610003A3
tissue: pancreas
agent: Thyroid Hormone (TH)
time: 24 hours
|
| 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, T3 exposure for 24 hours
|
| 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 |
|
