ABSTRACT: Cells: ACH-2, and A3.01, were obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH. ACH-2, is a chronically infected cell line harboring HIV-1 LAV strain, while A3.01 is the corresponding parental uninfected cell line. Cells were grown in RPMI-1640 (Invitrogen, San Diego, CA) with 10% fetal bovine serum (FBS, Invitrogen), 5% penicillin-streptomycin (Invitrogen), and 2mM glutamine (Invitrogen). Cells were maintained at a concentration of 1x10e6 cells/ml in T-175 flasks. Cell concentrations and cell viability were monitored throughout the experiment at all time points studied. Cells were induced by addition of 20 ng/mL of phorbol myristyl acetate (PMA or TPA, Sigma, St Louis, MO) for one hour, after which the cells were washed with phosphate buffered saline (PBS). Cells were harvested by centrifugation at 1000 rpm for 10 minutes, at the following times after induction, 0.5, 3, 6, 8, 12, 18, 24, 48, 72, and 96 hour(s). HIV-infected and uninfected cells maintained and harvested in parallel with the PMA-treated cells, but not induced with PMA ( No PMA)were also harvested. Harvested cells were washed thrice with ice-cold PBS to remove media; cell pellets were snap frozen using ethanol-dry ice mixture and stored at (80°C for subsequent RNA extraction. Total RNA Extraction: Total RNA was extracted using RNEasy Midiprep Kits per manufacturer's protocol (Qiagen, Valencia, CA). RNA concentrations and purity were measured by spectrophotometry, RNA quality (absence of RNA degradation) was assessed by gel electrophoresis. RNA concentration was adjusted to the levels required for subsequent microarray experiment protocols by concentration in a SpeedVac (Savant Instruments, Holbrook, CA). RNA samples (6-7 µg/µL) were stored in 100 µL TE buffer at -80°C. Microarray Studies: Total RNA obtained from induced chronically infected and corresponding uninfected parental cells were used for microarray experiments. For each time point, RNA from the induced chronically infected ACH-2 cells and RNA from the corresponding induced, uninfected A3.01 cells were compared to minimize effects due to PMA induction. Microarrays were obtained from the National Cancer Institute Microarray Facility, Advanced Technology Center (Gaithersburg, MD). The microarrays (Hs. UniGem2) contained 10,368 cDNA spots on each glass slide. The cDNAs were selected for spotting on the slides based on their known or probable involvement in oncogenesis, signal transduction, apoptosis, immune function, inflammatory pathways, cellular transport, transcription, protein translation and other important cellular functions. A number of expressed sequence tags (ESTs) from unknown genes homologous to known genes and cDNAs encoding housekeeping genes were also included in these gene sets. For each time point, 50 µg of total RNA from PMA induced ACH-2 cells and 70 µg of total RNA from PMA induced A3.01 cells was labeled with Cy-3-dUTP and Cy-5-dUTP respectively. Higher amounts of RNA were used for Cy-5 labeling to minimize the disparities in dye incorporation. Each sample of RNA from PMA-induced, infected cells from a particular time point was compared with RNA from the corresponding PMA-induced, uninfected cells from the same time point for subsequent hybridization to the same array to ensure accurate comparisons and to eliminate inter-array variability. Following reverse transcription, the labeled cDNAs were then combined and purified using MicroCon YM-30 (Millipore, Bedford, MA) spin column filters, to remove any unincorporated nucleotides. 8-10 µg each of Cot-1 DNA, (Boehringer Mannheim, Indianapolis, IN), yeast tRNA (Sigma) and polyA (Amersham Biosciences) were added to the reaction mixture and heated at 100°C for 1 minute. Hybridization of the labeled cDNA to the microarray was carried out at 65°C overnight, followed by washes with 1X SSC, 0.2X SSC and 0.05X SSC respectively. The slides were dried by centrifugation at 1000 rpm for 3 minutes and then scanned as described below. Microarray Scanning and Data Analysis: The slides were scanned using an Axon GenePix 4000 scanner (Axon Instruments, Union City, CA). The photomultiplier tube values (PMT) were adjusted to obtain equivalent intensities at both wavelengths used, 635 nm and 532 nm for the Cy5 and Cy3 channels respectively. Image analysis was performed using GenePix analysis software (Axon Instruments) and data analysis was performed using the microArray Database (mAdb) system hosted by the Center for Information Technology and Center for Cancer Research at NIH (http://nciarray.nci.nih.gov/). Keywords = HIV Keywords = latency
