Total RNA extracted using PAXgene blood RNA Kid (Qiagen, France) following manufacturer's instructions
Label
Cy3
Label protocol
5 µg ofRNA from both blood and reference samples were reverse transcribed to cDNAand fluorescent-labelled by Cy3 and Cy5, respectively, using the ChipShotTM Direct Labeling System (Promega, Charbonnieres, France). The CyDye-labelled cDNA were purified usingChipShotTMMembrane Clean-Up System (Promega, USA), and 750 ng each of Cy3-labeled and Cy5-labeled cDNA targets were combined for slide hybridization with a freshly prepared solution of 1% BSA, 2 x SSC, 0.1% SDS over 30 min at 50°C. After automatic washing according to manufacturer’sinstructions, the slides were hybridized for 17h at 60ºC.
reference: pooling of total RNAs from different porcine tissues
Extracted molecule
total RNA
Extraction protocol
Total RNA extracted using PAXgene blood RNA Kid (Qiagen, France) following manufacturer's instructions
Label
Cy5
Label protocol
5 µg ofRNA from both blood and reference samples were reverse transcribed to cDNAand fluorescent-labelled by Cy3 and Cy5, respectively, using the ChipShotTM Direct Labeling System (Promega, Charbonnieres, France). The CyDye-labelled cDNA were purified usingChipShotTMMembrane Clean-Up System (Promega, USA), and 750 ng each of Cy3-labeled and Cy5-labeled cDNA targets were combined for slide hybridization with a freshly prepared solution of 1% BSA, 2 x SSC, 0.1% SDS over 30 min at 50°C. After automatic washing according to manufacturer’sinstructions, the slides were hybridized for 17h at 60ºC.
Hybridization protocol
Oligoarray control targets and hybridization buffer (Agilent In Situ Hybridization Kit Plus) were added, and samples were applied to microarrays enclosed in Agilent SureHyb-enabled hybridization chambers. After hybridization, slides were washed sequential
Scan protocol
Detection of the fluorescence signals was made with a laser scanner (GenePix 4000A from Axon Instruments, CA) keeping a constant PMT gain fro each channel. The images were analysed with GenePixTM Pro 6.0 software (Axon instruments, Inc., Union City, CA).
Description
13890595 alternative to: GSM1098821
Data processing
All microarray analysis, including pre-processing, normalization and statistical analysis was carried out using Bioconductor packages in R programming language (version 2.14). The centring was preformed by “Lowes fitness”to take into account the intensity dependence of the fluorescence bias.
