Three batches of 100 blastocysts were collected for dormant and activated blastocysts respectively, and mRNA was extracted from each batch using a Quickprep micro poly-A RNA Extraction Kit (Amersham Biosciences). mRNA aliquots equivalent to 24 blastocysts from each mRNA subset were labeled with Cy3 dyes by two-round linear amplification labeling reactions for cRNA targets using Fluorescent Linear Amplification Kits (Agilent Technologies). The quality and size distribution of targets were determined by RNA 6000 Nano Lab-on-chip Assay (Agilent), and quantitation was determined using a microscale spectrophotometer (NanoDrop).
Label
Cy3
Label protocol
Three batches of 100 blastocysts were collected for dormant and activated blastocysts respectively, and mRNA was extracted from each batch using a Quickprep micro poly-A RNA Extraction Kit (Amersham Biosciences). mRNA aliquots equivalent to 24 blastocysts from each mRNA subset were labeled with Cy3 dyes by two-round linear amplification labeling reactions for cRNA targets using Fluorescent Linear Amplification Kits (Agilent Technologies). Briefly, mRNA was used to synthesize double-stranded cDNA with moloney murine leukemia virus (MMLV) reverse transcriptase in a reaction scaled down to a total volume of 4 ul, with half the standard T7-oligo-dT primer concentration and 125 ng/ul ofT4gp32 single-stranded DNA-binding protein (United States Biochemical, Cleveland, OH). Linear amplification (in vitro transcription) was performed in a total volume of 16 ul, with half the standard NTP concentration and no labeled CTP. For the second round of amplification, the product of the first reaction was divided in half, and labeled using the manufacturers standard protocol, with the addition of T4gp32 in the cDNA synthesis reaction. Quality and size distribution of targets was determined by an RNA 6000 Nano Lab-on-chip Assay (Agilent Technologies), and quantitation was determined using a NanoDrop microscale spectrophotometer (NanoDrop, Wilmington, DE). cRNA targets were assembled into hybridizations on the NIA 22K 60-mer oligo microarray (manufactured by Agilent Technologies)
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
activated blastocysts
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.
Global gene expression analysis identifies molecular pathways distinguishing blastocyst dormancy and activation
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 6 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
