Endometrial tissue was homogenised in Qiagen buffer RLT (QIAGEN GmbH, QIAGEN, Hilden, Germany) using a TissueLyser II (Qiagen). Approximately 30 mg of tissue was homogenized for 2 minutes with three ball bearings of 1/8 inch diameter (Farrell Bearings, Hamilton, NZ) in a tube containing RLT lysis buffer (Qiagen). Total RNA was extracted using a Qiagen RNeasy kit (QIAGEN). All samples were DNase treated using the Ambion DNA-free kit (Ambion, Austin, TX) according to the manufacturer's instructions. RNA quantity was determined by spectrophotometry in a Nanodrop ND-1000 (Nanodrop Technologies, Wilmington, DE). RNA integrity was checked using the Agilent 2100 Bioanalyzer with a RNA 6000 Nano LabChip kit (Agilent Technologies, Palo Alto, CA).
Label
Cy5
Label protocol
One hundred and fifty nanograms of RNA were amplified using the Agilent Low Input Quick Amp Labeling Kit, two color (Agilent, G2519F) to generate fluorescent complimentary RNA (cRNA) according to the manufacturer's instructions. cRNA quantity was measured by spectrophotometry using a ND-1000 (Nanodrop Technologies, Wilmington, DE).
tissue: caruncular and intercaruncular endometrium disease state: subclinical endometritis and control sample type: reference
Extracted molecule
total RNA
Extraction protocol
Endometrial tissue was homogenised in Qiagen buffer RLT (QIAGEN GmbH, QIAGEN, Hilden, Germany) using a TissueLyser II (Qiagen). Approximately 30 mg of tissue was homogenized for 2 minutes with three ball bearings of 1/8 inch diameter (Farrell Bearings, Hamilton, NZ) in a tube containing RLT lysis buffer (Qiagen). Total RNA was extracted using a Qiagen RNeasy kit (QIAGEN). All samples were DNase treated using the Ambion DNA-free kit (Ambion, Austin, TX) according to the manufacturer's instructions. RNA quantity was determined by spectrophotometry in a Nanodrop ND-1000 (Nanodrop Technologies, Wilmington, DE). RNA integrity was checked using the Agilent 2100 Bioanalyzer with a RNA 6000 Nano LabChip kit (Agilent Technologies, Palo Alto, CA).
Label
Cy3
Label protocol
One hundred and fifty nanograms of RNA were amplified using the Agilent Low Input Quick Amp Labeling Kit, two color (Agilent, G2519F) to generate fluorescent complimentary RNA (cRNA) according to the manufacturer's instructions. cRNA quantity was measured by spectrophotometry using a ND-1000 (Nanodrop Technologies, Wilmington, DE).
Hybridization protocol
Eight hundred and twenty five nanograms of Cy3- and Cy5-labeled cRNA were added to Agilent-023647 4x44k 60-mer oligonucleotide microarrays (G2519F 023647) and hybridized overnight at 65°C (17 hours). Slides were then washed with Agilent Gene Expression Wash Buffers 1 and 2 according to the manufacturer's instructions, inserted into a slide cover with ozone barrier, and scanned on an Agilent C scanner at 5µm.
Scan protocol
Slides were scanned on an Agilent C scanner at 5µm.
Description
En3 32
Data processing
Agilent Feature Extraction software version 10.7.3.1 was used to analyze the scanned Agilent microarray. The 24 scanned microarray image files were uploaded to the Feature Extraction software. Using a design file (023647), the Feature Extraction software locates features and converts the extracted data from each feature into a quantitative log2ratio. This software removes pixel outliers, performs statistical tests on the non-outlier pixels, subtracts background from features and flags any outlier features. The software was then used to perform LOWESS (locally weighted linear regression analysis) dye normalization and to calculate a p-value for each feature.
