embryonic stem cell 129/Ola Transgenic and Knock down
Biomaterial provider
Dr. Hitoshi Niwa, Laboratory for Pluripotent Cell Studies,RIKEN Center for Developmental Biology,2-2-3 Minatojima-minamimachi,Chu-o-ku, Kobe 6500047,Japan. E-mail: [email protected]
Extracted molecule
total RNA
Extraction protocol
TROZOL standard extraction method. The full protocol can be found in the Life Technologies CAT 15596. The following are the steps we used. 1.Homogenize tissue samples in 1 mL of TRIZOL Reagent per 50-100 mg of tissue using a glass-Teflon® or power homogenizer (Polytron, or Tekmar's TISSUMIZER® or equivalent). The sample volume should not exceed 10% of the volume of TRIZOL Reagent used for homogenization. 3.PHASE SEPARATION Incubate the homogenized samples for 5 minutes at 15 to 30°C 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 15 to 30°C for 2 to 3 minutes. Centrifuge the samples at no more than 12,000 x g for 15 minutes at 2 to 8°C. 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. 4. RNA PRECIPITATION Transfer the aqueous phase to a fresh tube, and save the organic phase if isolation of DNA or protein is desired. 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 15 to 30°C for 10 minutes and centrifuge at no more than 12,000 x g for 10 minutes at 2 to 8°C. The RNA precipitate, often invisible before centrifugation, forms a gel-like pellet on the side and bottom of the tube. 5. RNA WASH 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 2 to 8°C. 6. REDISSOLVING THE RNA At the end of the procedure, briefly dry the RNA pellet (ai
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).
The UMRR is provided in a solution of 70% ethanol and 0.1 M sodium acetate. Prepare the UMRR for use as follows: 1.Centrifuge the tube at 12,000 x g for 15 minutes at 4C. 2.Carefully remove the supernatant. 3.Wash the pellet in 70% ethanol. 4.Centrifuge the tube at 12,000 x g for 15 minutes at 4C. 5.Carefully remove the supernatant and air-dry the pellet at room temperature for 30 minutes to remove retained ethanol. 6.Resuspend the pellet in RNase-free water to the desired concentration.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from cultured cells using a Stratagene Absolutely RNA kit. DNase-treated total RNA samples from 11 cultured cell lines derived from embryo, embryo fibroblast, kidney, liver/hepatocyte, lung/alveolar macrophage, B-lymphocyte, T-lymphocyte (thymus), mammary gland, muscle myoblast, skin, and testis were pooled in equal parts
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. The manual can be found at https://www.chem.agilent.com/scripts/literaturePDF.asp?iWHID=34961, or through a WEB search on the manual title.
Scan protocol
Slides were scanned with an Agilent DNA Microarray Scanner model G2505-64120 at 100% PMT in both channels, with a scan resolution of 10um. A scan window of 61 x 21.6 mm was used, then images were cropped to size and saved in a modified two-color TIFF format. Images were examined visually for evidence of foreign debris or major failures. Agilent Microarray Scanner User Manual (6.3) http://www.chem.agilent.com/scripts/literaturePDF.asp?iWHID=33696
Description
Sox2-KO, Tet(-), 24hr
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.
Pluripotency governed by Sox2 via regulation of Oct3/4 expression in mouse embryonic stem cells
Data table header descriptions
ID_REF
Feature Number (FeatureNum)
VALUE
The normalized VALUE among all the arrays in the series. It is caculated 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 12 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
