RNA was extracted using the E.Z.N.A total RNA kit (VWR International, Mississauga, ON, CA). RNA quantity was measured using the NanoDrop ND2000 (Thermo Scientific) prior to DNase treatment for real-time PCR and RNA integrity was evaluated according to Agilent 2100 Bioanalyzer protocol (Agilent, Mississauga, ON, CAN).
Label
Cy3
Label protocol
Agilent one color protocol for Cy3
Hybridization protocol
Silurana tropicalis microarrays were manufactured with Agilent Sure Print Technology in Fall 2011. RNA labeling, microarray hybridization, and microarray analysis were performed according to Agilent’s One-Color Microarray-Gene Based Expression Analysis protocol (May 2010) using an Agilent kit (Agilent, Mississauga, ON, CAN). After hybridization for 17 hours, microarray slides were washed and scanned using Agilent Technologies DNA Microarray Scanner with Surescan High-Resolution Technology and raw expression data along with tif images were extracted by the Feature Extraction Software 10.7.3.1.
Scan protocol
After hybridization for 17 hours, microarray slides were washed and scanned using Agilent Technologies DNA Microarray Scanner with Surescan High-Resolution Technology and raw expression data along with tif images were extracted by the Feature Extraction Software 10.7.3.1.
Data processing
Normalized signal intensity provided here is LOESS Normalization (JMP Genomics, V6.0).
