Detection of pp71 RNA and protein in primary glioma specimens.

<p><b>A:</b> RNA was extracted from 20 different primary brain tissues, synthesized into cDNA, and amplified using pp71 and Rab14-specific PCR primers. RNA from U251 glioma cells mock infected or infected with HCMV Towne strain and commercially available RNA samples from normal fetal and adult human brain were used as controls. The N.C. negative control PCR contained water in place of cDNA. <b>B</b>: Several cDNA samples described in A were analyzed by TaqMan using primers and probes specific for the pp71 gene and normalized to Rab14. Copy number was determined using Ad169 viral DNA standard curve. <b>C</b>: Western blot analysis for pp71 from 10 different primary brain tissues. Lysates from normal human astrocytes and mock-infected or HCMV-infected U87 glioma cells were used as controls. <b>D</b>: Cells from 3 freshly resected GBM were sorted using CD133 labeled antibody and analyzed by RT-PCR for pp71 and Rab14. Negative control (NC) is PCR performed with water instead of cDNA. <b>E</b>: HCMV-infected primary GBM cell line (3832) was sorted using CD133 labeled antibody at 72 hours post-infection and analyzed by TaqMan for pp71 compared to a mock-treated control. The relative expression of pp71 normalized to Rab14 is displayed for 3 independent experiments, *p<0.05 by student t-test.