Figure 3. Detection of the ORF1-like expression in a PML brain tissue sample by RT-PCR.

(A) (Upper panel) Detection of a three-banding pattern in a PML patient tissue samples by RT-PCR. Initially, PML and non-PML tissue samples were obtained from the Manhattan HIV Brain Bank and total RNA isolated from those samples was subjected to one step RT-PCR as described in Materials and Methods using JCV 5′-primer (277-302) and JCV 3′-primer (780-752) (Mad-1 numbering). A new internal 3′-primer was used in this RT-PCR to amplify relatively short fragments and therefore resolve them well on the agarose gels. The hatched and filled arrow heads indicate the possible expression of ORF1 and perhaps additional ORFs respectively. The labeled arrow points to the expected spliced product. In lane 4, JCV Mad-1 DNA was used as positive control (+ Cont.) and in lane 5, water was used as a negative control (− Cont.) in PCR reactions. (Lower panel) Total RNA was also subjected to RT-PCR using human actin primers as a control: Forward primer: Human actin (354-375) 5′-CTACAATGAGCTGCGTGTGGC-3′ and Reverse primer: Human actin (624-603) 5′-CAGGTCCAGACGCAGGATGGC-3′. (B) Detection of JCV DNA in PML tissue samples. In parallel to the panel A, JCV DNA isolated from the same PML and non-PML brain tissue samples were also amplified by PCR as described in Materials and Methods, using the same primers described for panel A.