Agilent One-Color Microarray-Based Gene Expression (Hybridization) Version: 5.7 Agilent publication number: G4140-90040 URL: http://www.chem.agilent.com/Library/usermanuals/Public/G4140-90040_One-Color_GE_5.7.pdf This document describes Agilent's recommended procedures for the preparation and labeling of complex biological targets and hybridization, washing, scanning, and feature extraction of Agilent 60-mer oligonucleotide microarrays for microarray-based one-color gene expression analysis.
We normalized all samples simultaneously using a multiple-loess technique described in:
Sasik R, Woelk CH, Corbeil J., J Mol Endocrinol. 33:1-9 (2004).
Scanning of the agilent microarrays was performed through the standard agilent microarray scanner system protocol.
(Parameters: Scanning hardware = DNA Microarray Scanner BA [Agilent Technologies], Scanning software = Feature Extraction Software [Agilent])
Three different sample types were used: unwounded wild-type, puncture wild-type, and trypsin puncture wild-type stage 15-17 embryos. Unwounded wild-type stage 15-17 embryos were used as controls. Puncture wild-type embryos were wounded with 1mM HCl and dye and allowed to recover for either 30, 60, or 120 minutes at RT before RNA isolation. Trypsin puncture wild-type embryos were wounded with 2mg/mL trypsin soluble in 1mM HCl and dye and were allowed to recover for either 30, 60, or 120 minutes before RNA isolation.
Parental wild-type (w1118) flies were raised under standard lab conditions, and the laid embryos were allowed to develop at 25C for approximately 14-16 hours. Gut morphology was used as a guide to ensure proper staging.
(Parameters: time unit = seconds, temperature unit = C)
Whole embryos (either wounded or unwounded) were collected and stored frozen in Trizol (Invitrogen). For each replicate, approximately 500 whole embryos were ground in Trizol in microfuge tube with a pestle according to the manufacturer's recommendations. For each sample, 50 micrograms of RNA was further cleaned using the Qiagen RNeasy miniprep kit, according to the manufacturer's recommendations. Sample integrity was assayed using RT-PCR and BioAnalyzer (Agilent) analysis.
(Parameters: Extracted product = total_RNA, Amplification = none)