heparinized blood samples, peripheral blood mononuclear cells isolated with Ficoll density gradients, cells lysed in Trizol and stored at -80
Extracted molecule
total RNA
Extraction protocol
Trizol followed by Qiagen Rneasy columns
Label
biotin
Label protocol
RNA expression was profiled on Illumina (San Diego, CA) beadchip humanRef-8 v3.0 microarrays at the UTHSC-H Quantitative Genomics Core Laboratory. Two hundred ng of total RNA were amplified and purified using Illumina TotalPrep RNA Amplification Kit (Ambion, Austin, TX) following kit instructions. Amplified cRNA was subsequently purified and concentration was measured by NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, DE). An aliquot of 750 ng was loaded onto the microarray, hybridized at 58°C in an Illumina Hybridization Oven for 17 hours, washed and incubated with streptavidin-Cy3 to detect the biotin-labeled cRNA. Arrays were dried and scanned with the Illumina BeadArray Reader. Each chip contains 8 separate microarrays, and the samples were run on 5 separate chips in 4 different experiments with the stable and relapse specimen for each patient on the same chip. Data were extracted, background subtracted, and normalized by quantiles using Illumina BeadStudio 3.2, and then exported to a worksheet. These files for all experiments were combined in a single worksheet, and data for non-expressed transcripts were removed. The edited worksheet was then analyzed using BRB-ArrayTools 3.8.0 and Ingenuity (version 7).
Hybridization protocol
standard Illumina protocol
Scan protocol
standard Illumina protocol
Description
n/a
Data processing
background subtracted, normalized to quantiles in BeadStudio, paired comparison of normalized data in BRB
