Specific silencing of hSpt5 expression by RNAi. (A) hSpt5 mRNA is 3261 nucleotides in length. siRNA targeting sequence for hSpt5 was selected from position 407 to 427 relative to the start codon. As a specific control, mutant siRNA containing 2 nucleotide mismatches (underline) between the target mRNA and the antisense of siRNA at the hypothetical cleavage sites of the mRNA was generated. (B) Evaluation of specific hSpt5 siRNA activity by RT-PCR. Total cellular mRNA was prepared from HeLa cells transfected without siRNA or with hSpt5 duplex or control siRNAs and was followed by RT-PCR, as described in Material and Methods. Each RT-PCR reaction included 100 ng total cellular mRNA, gene-specific primer sets for hSpt5 and hCycT1 amplification (0.5 μM for each primer), 200 μM dNTP, 1.2 mM MgSO4 and 1U of RT/platinum Taq mix. Primer sets for hSpt5 produced 2.6 kb products while hCycT1 produced 1.8 kb products. RT-PCR products were resolved on a 1% agarose gel and viewed by ethidium bromide staining. RT-PCR products are shown from cells that were not transfected with siRNA (lane 1), or cells transfected with single-stranded antisense hSpt5 siRNA (hSpt5 (AS), lane 2), hSpt5 duplex siRNA (hSpt5 (DS), lane 3), or mismatch hSpt5 duplex siRNA (hSpt5-mm (DS), lane 4). Lane M is a marker lane. (C) Analysis of specific hSpt5 siRNA activity by western blotting. Cell lysates were prepared from HeLa cells mock-transfected without siRNA (lane 1), or transfected with single-stranded antisense hSpt5 siRNA (hSpt5 (AS), lane 2), hSpt5 duplex siRNA (hSpt5 (DS), lane 3), or mismatch hSpt5 duplex siRNA (hSpt5-mm (DS), lane 4). Cell lysates were analyzed by 10% SDS-PAGE. Protein contents were detected by immunoblotting assay with antibodies against hSpt5 (top panel) and hCycT1 (lower panel).