Customized rapid subtraction hybridization (RaSH) gene microarrays identify overlapping expression changes in human fetal astrocytes resulting from human immunodeficiency virus-1 infection or tumor necrosis factor-α treatment

Abstract

Genes displaying altered expression as a function of human immunodeficiency virus (HIV)-1 infection of cultured primary human fetal astrocytes (PHFA) were previously identified using a rapid subtraction hybridization (RaSH) method. This scheme identified both known and novel genes displaying elevated expression, astrocyte elevated genes ( AEG), and decreased expression, astrocyte suppressed genes ( ASG), in PHFA as a consequence of infection with HIV-1 or treatment with HIV-1 envelope glycoprotein (gp120). RaSH also identified both known and novel genes displaying enhanced ( HR) or reduced ( HS) expression in HIV-1 resistant versus HIV-1 susceptible human T-cell clones. In the present study, a customized microarray approach employing these RaSH-derived genes was used to distinguish overlapping gene expression changes occurring in PHFA as a function of treatment with HIV-1 and the neurotoxic agent tumor necrosis factor (TNF)-α. RaSH cDNAs were spotted (microarrayed) on nylon membranes and probed with temporally isolated reverse transcribed cDNAs from HIV-1-infected and TNF-α-treated PHFA. This strategy identified genes displaying parallel changes after TNF-α treatment as observed following HIV-1 infection. Confirmation of genuine differential expression was achieved by Northern blotting. These studies document that TNF-α can induce a set of corresponding changes in specific AEGs and ASGs as does HIV-1 infection in PHFA. Furthermore, this customized microarray approach with RaSH-derived clones represents an efficient and sensitive methodology for elucidating molecular changes in PHFA occurring as a consequence of treatment with pharmacological agents affecting astrocyte physiology.