Universal Human Reference RNA (catalog # 740000) was purchased from Stratagene (LaJolla, CA, USA)
Treatment protocol
none
Growth protocol
none
Extracted molecule
total RNA
Extraction protocol
Amplified cDNA was prepared using the WT-Ovation™ Pico RNA Amplification System (NuGen product #3300; Santa Clara, CA, USA) starting with 50 ng of each total RNA sample (A, B, C, D), as described by the manufacturer. Briefly, the RNA is synthesized into double-stranded cDNA using a SPIA™ DNA/RNA primer. The DNA portion of the primer binds poly (A) sequence or randomly across the transcript, and the RNA portion of the primer is incorporated into an unique DNA/RNA heteroduplex at one end of the cDNA. RNase H degrades the RNA portion of the DNA/RNA heteroduplex allowing for additional SPIA™ primer to bind and initiate replication by DNA polymerase. The process of SPIA™ DNA/RNA primer binding, DNA replication, strand displacement and RNA cleavage is repeated resulting in isothermal linear amplification of the cDNA. On average, the cDNA yield from 50 ng of high quality total RNA ranges from 6 to 10 micrograms.
Label
Biotin
Label protocol
Fragmentation and biotin-labeling of the amplified cDNA was performed using the FL-Ovation™ cDNA Biotin Module V2 (NuGen product #4200; Santa Clara, CA, USA) as per manufacturer’s instructions.
Hybridization protocol
Standard hybridization methods of the labeled samples to the Affymetrix-formatted SpliceArrayTM were used following the manufacturer’s recommendations for the 49-format (Affymetrix - Expression analysis technical manual). The arrays were stained and washed using the Affymetrix FS450-0001 fluidics protocol.
Scan protocol
Arrays were scanned using Affymetrix GeneChip® Scanner 3000 7G. DAT and CEL images were visually inspected for anomalies and accurate grid placement.
Description
none
Data processing
Data processing was performed using Partek® software (http://www.partek.com/). 12 .CEL files were imported into Partek® software. All array data was pre-processed which included probe level RMA background correction, quantile normalization across all arrays, and Log2 transformation followed by median polish to summarize probes to obtain the overall score for each probe set.
