Fresh-frozen samples were obtained from 14 patients including 7 primary central nervous system diffuse large B cell lymphoma and 7 normal lymph nodes.
Extracted molecule
total RNA
Extraction protocol
RNA was extracted from 14 fresh-frozen tumor samples using standard proteinase K/RNAse treatment and phenol-chloroform extraction method. RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
Label
Cy3
Label protocol
0.5 ug of total RNA was amplified and labeled using Agilent Quick Amp Labeling Kit. After labeling, cRNA was purified with Qiagen’s RNeasy Mini-spin columns (QIAGEN, Germantown, MD) and quantified using NanoDrop ND-1000 UV-VIS Spectrophotometer for cyanine 3 dye concentration, RNA absorbance ratio (260 nm/280 nm), and cRNA concentration.
Hybridization protocol
Cyanine 3-labled linearly amplified cRNA was mixed with 10X blocking agent, nuclease-free water, and 25X fragmentation buffer provided in the Agilent Gene Expression Hybridization Kit (Agilent Technologies), and incubated for 30 min at 60oC to fragment RNA. After adding 2X GEx hybridization buffer Hi-RPM, the amplified fragmented RNA was hybridized on a 4 x 44K Whole Human Genome Oligo Microarray Chip (Agilent Technologies) containing 44,000 cDNA clones using a SureHyb DNA Microarray Hybridization Chamber in a DNA Microarray Hybridization Oven (Agilent Technologies) at 10 rpm, 65oC for 17 hours.
Scan protocol
Slides were scanned with an Agilent DNA microarray scanner (Agilent Microarray Scanner–G2565BA, Agilent Technologies).
Description
Gene expression
Data processing
Feature Extraction Software v9 (Agilent Technologies) was used to extract and analyze the signals. To validate the amplification process, amplified and unamplified RNAs were labeled, hybridized and compared.
