development stage: tadpole [Nieuwkoop and Faber stage (NF) 62] tissue: pancreas individual identifier: 251321410051 agent: Thyroid Hormone (TH)
Biomaterial provider
Donald Brown,Carnegie Institution,3520 San Martin Dr,Baltimore,MD,21218 E-mail: brown@ciwemb.edu
Treatment protocol
Xenopus tadpoles (NF52, NF62, and NF66) 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 and liver 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).
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
62-Pan
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 9 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
