Cells: ACH-2, A3.01, J1.1, and U1 cells were obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH. U-937 cells were obtained from American Type Culture Collection (Manassas, VA). ACH-2, J1.1 and U1 are chronically infected cell lines harboring HIV-1 LAV strain, while A3.01, Jurkat, and U-937 are the corresponding parental uninfected cell lines. 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. Cells were harvested by centrifugation at 1000 rpm for 10 minutes. 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. In order to compare expression profiles of chronically infected cell lines, ACH-2, U1, and J1.1 and their uninfected parental cell lines, A3.01, U-937 and Jurkat cells respectively, were grown under identical conditions but in presence of AZT (250 nM) in growth media and cells were harvested as described above. In these studies, no inducing agent was used in either chronically infected or uninfected parental cell lines so as to study changes in cellular gene expression cells latently infected with HIV and uninfected cells. 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 chronically infected and corresponding uninfected parental cells were used for microarray experiments. For each cell line, RNA from the chronically infected cells (ACH-2, J1.1 or U1)and RNA from the corresponding induced, uninfected cells (A3.01 , Jurkat or U937) were compared on the same array. 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 array, 50 µg of total RNA from chronically infected cells and 70 µg of total RNA from corresponding uninfected 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. 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 Keywords: other
Less...
More...