cell line: T157 tissue: Uveal melanoma derivative cell line gender: female age: 33 years
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated using the RNeasy Mini Kit (QIAGEN, Toronto, ON) from the uveal melanoma cell lines T97, T98, T108, T111, T128, T131, T132, T142, T143, T151, T157 cultured from the primary tumors T97, T98, T108, T111, T128, T131, T132, T142, T143, T151, T157 of patients diagnosed with uveal melanoma .
Label
Cy3
Label protocol
Cyanine-3 (Cy3) labeled cRNA was prepared from 50 ng RNA using the One-Color LowInput QuickAmp Labeling Kit One-Color (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Toronto, ON). Dye incorporation and cRNA yield were checked with the Nanophotometer pearl(Montréal Biotech).
Hybridization protocol
600 ng of Cy3-labelled cRNA (specific activity >8.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25 ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 25 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole SurePrint G3 Human GE 8x60K (G4851A) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately.
Scan protocol
Slides were scanned immediately after washing on the Agilent SureScanner (G4900DA) using one color scan setting for 1x60k array slides (Scan resolution 3um, Dye channel is set to Green and Green PMT is set to 100%).
Data processing
The scanned images were analyzed with Feature Extraction Software 10,7 (Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
Transcription of the human 5-Hydroxytryptamine receptor 2B (HTR2B) gene is under the regulatory influence of the transcription factors NFI and RUNX1 in human uveal melanoma
