tissue: peripheral blood subject: Control rna prep: PAXgene molecule: total RNA
Treatment protocol
Peripheral blood mononuclear cell isolation: Peripheral blood from sickle-cell patients, and healthy African-American volunteers, was collected into Vacutainer cell preparation tube (CPT) with sodium citrate and Ficoll (Becton Dickinson, Franklin Lakes, NJ). Purified peripheral blood mononuclear cell (PBMC) suspensions were resuspended in buffer RLT (700-1000 µL per 107 cells) and passed through Qiashredder columns (Qiagen, Valencia, CA) then stored at –70° C.
Extracted molecule
total RNA
Extraction protocol
Peripheral blood mononuclear cell RNA isolation: Total RNA was extracted from peripheral blood mononuclear cells using RNeasy Mini Kit (Qiagen). Isolation of RNA from whole blood specimens: Blood Specimen (2.5ml) collected in PAXgene™ tubes from each subject was incubated at room temperature for 4h for RNA stabilization and then stored at − 80 °C. RNA was extracted from whole blood using the PAXgene™ Blood RNA System Kit following the manufacturer's guidelines. Briefly, samples were removed from -80°C and incubated at room temperature for 2 hours to ensure complete lysis. Following lysis, the tubes were centrifuged for 10 min at 5,000 × g the supernatant was discarded and 500 μL of RNase-free water added to the pellet. The tube was vortexed thoroughly to re-suspend the pellet, centrifuged for 10 min at 5000 × g and the entire supernatant was discarded. The pellet was re-suspended in 360 μL of buffer BR1 by vortexing and further purification of RNA was done following the manufacturer's protocol with on-column DNase digestion. Depletion of Globin Transcripts: Globin mRNA was depleted from a portion of each total RNA sample isolated from PAXgene tubes using the GLOBINclear™-Human kit (Ambion, Austin, TX). In brief 4 µg of total RNA from human whole blood was mixed with a biotinylated Capture Oligo Mix in hybridization buffer. The mixture was incubated for 15 minutes to allow the biotinylated oligonucleotides to hybridize with the globin mRNA species. Streptavidin magnetic Beads were then added, to capture the globin mRNA and the magnetic beads were then pulled to the side of the tube with a magnet and the RNA, depleted of the globin mRNA, was transferred to a fresh tube. The RNA was further purified using a rapid magnetic bead-based purification method.
Label
biotin
Label protocol
The cRNA labeling and hybridizations were performed according to protocols from Affymetrix Inc. (Santa Clara, CA). Briefly, 2 µg of total RNA from PBMC (n=10, 5 controls and 5 SCD), whole blood (PAX) (n=10, 5 controls and 5 SCD) and Globin depleted (PAX-GR) (n=10, 5 controls and 5 SCD) samples was converted to double-stranded cDNA with a T7-(dT) 24 primer. The cDNA was in vitro transcribed to biotinylated complementary RNA (cRNA) by incorporating biotin-CTP and biotin-UTP using affymetrix IVT labeling kit.
Hybridization protocol
Twenty µg of biotin-labeled RNA was fragmented to ~200 bp size by incubating in fragmentation buffer containing 200 mM Tris-acetate pH 8.2, 500 mM potassium acetate and 500 mM magnesium acetate for 35 minutes at 94ºC prior to hybridization. Fragmented RNA was assessed for relative length on Agilent 2100 bioanalyzer, and hybridized to Affymetrix HG-U133 plus 2.0 chips for 16 hours, washed, and stained on an Affymetrix fluidics station.
Scan protocol
Arrays were scanned using an Affymetrix genechip scanner 3000.
Description
standard labeling and hybridization procedure
Data processing
Affymetrix GCOS version 1.4 was used to calculate the signal intensity and the percent present calls on the hybridized Affymetrix chip. The signal intensity values obtained for probe sets in the microarrays were transformed using an adaptive variance-stabilizing, quantile-normalizing transformation termed “S10” (Munson, P.J., GeneLogic Workshop of Low Level Analysis of Affymetrix GeneChip Data, 2001, software available at http://abs.cit.nih.gov/geneexpression.html.).
