7 protocols
ID_REF = VALUE = Normalized signal intensity gSigEval =
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2565CA) using one color scan setting for 8x60k array slides (Scan Area 61x21.6 mm, Scan resolution 3um, Dye channel is set to Green PMT is set to 100%).
The scanned images were analyzed with Feature Extraction Software 10.10 (Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities (gScale signal). the gSigEval column shows the results of Signal Evaluation; 2 for transcripts detected,1 for transcripts detected with low level (indicating the signal intensity data might be affected), 0 for transcripts that were not detected.
2.0 ug of Cy3-labelled cDNA in a reaction volume of 25 ul containing 1x Agilent blocking agent following the manufacturers instructions. 25 ul of 2x Agilent hybridization buffer was added to the mixture and hybridized to Agilent SurePrint G3 Mouse GE 8x60K Microarrays for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed for 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and for 1 minute at 37°C with GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
Cyanine-3 (Cy3) labeled cDNA was prepared from 6.1ng Total RNA using the Ovation® Pico WTA System V2 (NuGEN) according to the manufacturer's instructions, followed by DNA Clean & Concentrator-25 purification (Zymo Research). Dye incorporation and cDNA yield were checked with the NanoDrop ND-2000 Spectrophotometer.
RNA was prepared using Rneasy Micro Kit(50) (QIAGEN) following the manufacturer's recommendations.
Freshly isolated lympho-hematopoietic progenitor cells from fetal liver