One color microarray based gene expression analysis low input quick amp labeling protocol

Labelling was done as recommended by Agilent Technologies using the One-Color Microarray-Based Gene Expression Analysis Low input Quick Amp Labeling. The input was 200 ng of total RNA and 600 ng of labelled cRNA was used on the 8 pack array.
Hybridization was performed as described in the One-Color Microarray-Based Gene Expression Analysis Low input Quick Amp Labeling protocol from Agilent in the hybridization oven (G2545A hybridization Oven Agilent Technologies). The hybridization temperature was 65°C with rotation speed 10 rpm for 17 hours. After 17 hours the arrays were washed as described in the One-Color Microarray-Based Gene Expression Analysis Low input Quick Amp Labelling protocol from Agilent.
The porcine Agilent microarray slides, G2519F Sus scrofa (035953; V2∶026440), harbouring 43,803 probes, were used and scanned using the DNA microarray scanner with Surescan high resolution Technology (Agilent Technologies). Agilent Scan Control with resolution of 5 µ, 16 bits and PMT of 100%. Feature extraction was performed using protocol 10.7.3.1 (v10.7) for 1 colour gene expression.

Total RNA was extracted using the AllPrep DNA/RNA Micro kit (Qiagen, Hilden, Germany; cat. no. 80284) according to the manufacturer’s instructions. RNA concentration and integrity were evaluated using the Agilent RNA 6000 Nano Kit (Agilent Technologies, Santa Clara, CA, USA; cat. no. 5067-1511) on an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Microarrays were then further computationally processed as described in One-Color Microarray-Based Gene Expression Analysis Low Input Quick Amp Labeling protocol (Agilent Technologies, Santa Clara, CA, USA).