7 protocols
Title: Affymetrix CEL analysis. Description:
Title: Affymetrix Generic Hybridization. Description:
RNA was isolated using the RNeasy Plant Mini Protocol (QIAGEN, Clifton Hill, Australia) and the quality of the RNA was assessed using the Agilent Bioanalyzer (Agilent Technologies, Palo Alto, CA) where spectrophotometric analysis was carried out to determine the A260 to A280 ratio and the concentration. For qRT-PCR, cDNA synthesis was carried out using the Biorad (Perth) iScript cDNA synthesis kit according to manufacturers instructions. For arrays, the cRNA was prepared from 3 ug of total RNA almost exactly as described in the Affymetrix GeneChip Expression Analysis Technical Manual.
Blood samples (9 mL) were collected in EDTA-tubes at baseline and at week 2 of cART. The EDTA blood tubes were centrifuged at 1500 rpm for 10 minutes at room temperature. Peripheral blood mononuclear cells (PBMCs) were isolated from the blood fraction by Ficoll-Paque Plus gradient separation (GE Healthcare, Little Chalfont, UK). PBMCs were washed once with 50 mL HBSS (Gibco-Invitrogen, Carlsbad, USA) and once with 50 mL RPMI supplemented (Lonza BioWitthaker, Walkersville, MD, USA) with 100U/ml penicillin and 100 mg/mL streptomycin. PBMC pellets were resuspended in cold cryomedium (containing 40% RPMI, 10% DMSO and 50% human serum) and transferred to cryotubes. PBMCs were cryopreserved in liquid nitrogen prior further processing.
(Parameters: start time = 2.0, time unit = weeks, min temperature = 20, temperature unit = C, media = Cryomedium )
The RMA procedure was followed to normalize data within arrays (probeset summarization, background correction and log2-transformation) and between arrays (quantile normalization). The RMA-normalized expression values as obtained with the xps 1.8.0 package of BioConductor (www.bioconductor.org). About 90% of the identifiers found in the normalised data file are ?ǣtranscript cluster Ids?ÇØ in the NetAffx annotation file (?ǣHuGene-1_1-st-v1.na30.1.hg19.probeset?ÇØ annotation file from Affymetrix Technical Documentation, release 30). The remaining ~10% of identifiers are ?ǣprobeset_ids?ÇØ in the annotation file. Both types of identifiers can be found in the ArrayExpress array design file A-MEXP-2212.
Total RNA and protein fractions were isolated from Trizol lysates by chloroform extraction and isopropanol precipitation respectively, as per the manufacturer??Æs recommendations. Due to the low number of monocytes obtained from very limited PBMC samples, the RNA extraction required addition of RNase-free glycogen (Invitrogen, Carlsbad, CA, USA) as RNA carrier to enhance RNA precipitation and avoid the loss of samples. Using the Ambion WT Expression Kit, per sample, an amount of 100 ng of total RNA spiked with bacterial poly-A RNA positive controls (Affymetrix) was converted to double stranded cDNA in a reverse transcription reaction. Next the sample was converted and amplified to antisense cRNA in an in vitro transcription reaction which was subsequently converted to single stranded sense cDNA.
(Parameters: Extracted product = total_RNA, Amplification = RNA polymerases)
Monocytes were isolated from the cryopreserved PBMC samples using positive magnetic selection on CD14 (Miltenyi-Biotec, Bergisch Gladbach, Germany) according to the manufacturer??Æs instruction. Purified monocytes were immediately lysed in 1 mL Trizol (Invitrogen, Carlsbad, CA, USA) and lysates were stored at -80???C.