tissue: Peripheral blood NK cells in pregnancy trimester: 3rd
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted with RNAiso reagent (TaKaRa, Shiga, Japan) according to the manufacturer’s instructions. The integrity of the total RNA was determined using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).
Label
Cy3
Label protocol
Cyanine-3 (Cy3) labeled cRNA was prepared from 20 ng total RNA using a Low-Input QuickAmp Labeling Kit, One-Color (Agilent Technologies, Santa Clara, CA) according to the manufacturer's instructions, followed by RNAeasy column purification(QIAGEN,Valencia, CA). Dye incorporation, quality and cRNA yield were checked with the NanoDrop 2000 (Thermo Scientific).
Hybridization protocol
1.65 μg of Cy3-labelled cRNA (specific activity >6.0 pmol Cy3/μg cRNA) was fragmented at 60°C for 30 min in a reaction volume of 55 μl containing 2.2 μl of 25x Agilent fragmentation buffer, 11 μl of 10x Agilent blocking agent and 41.8 μl Nuclease-free water following the manufacturers instructions. On completion of the fragmentation reaction, 55 μl of 2x GEx hybridization buffer HI-RPM was added to the fragmentation mixture and hybridized to Agilent Human GE 4×44 K, v2 Microarray for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 min at room temperature with Wash Buffer 1 (Agilent) and 1 min with 37°C GE Wash buffer 2 (Agilent).
Scan protocol
The slide was scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505C) using one color scan setting for 4x44 K array slides (Scan Area 61x21.6 mm, Scan resolution 5 μm, Dye channel is set to Green and Green PMT is set to 100%).
Data processing
The scanned images were analyzed with Feature Extraction Software 10.7 (Agilent) using default parameters (protocol GE1_107_Sep09 and Grid: 026652_D_F_20110325).
