Figure 5.

Northern blots of small RNAs extracted from Igl and PATMK transfectants. To test if the U6 promoter was driving hairpin expression, shRNA transfectants (PATMK (3552–3580), PATMK (2273–2301), PATMK (3552–3580 scrambled) [39], Igl (1198–1226), Igl (2412–2440), and Igl (2777–2805) were selected with 30 μg/ml hygromycin for 48 hours before harvesting. HM1:IMSS non-transfected amebae were included as negative controls. Small RNAs were extracted using the mirVana™ miRNA Isolation Kit (Ambion) (Applied Biosystems/Ambion, Austin, TX, USA). Fifty μg small RNA were loaded per lane on a 12% denaturing acrylamide gel and transferred to membrane. rRNA bands were analyzed to ensure equal RNA loading. Oligo probes matching to the sense and antisense strands of the hairpins were end-labeled with 32P and were hybridized with each corresponding sample blot overnight at 37°C overnight, washed with low and medium stringency conditions, and exposed overnight to film. Note the two product sizes, which may correspond to the unprocessed hairpin (~60–70 nucleotides) (blue arrows) and the processed siRNA products (~30 nucleotides) (red arrows).

Linford et al. BMC Microbiology 2009 9:38   doi:10.1186/1471-2180-9-38
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